Some Structural Characteristics of the Chloroplasts in the “Glucose-bleaching” and Re-greening Cells of Chlorella protothecoides1)

Biochem. Physiol. Pflanzen 168, S. 533 -542 (1975)

Some Structural Characteristics of the Chloroplasts in the "Glucose-bleaching" and Re-greening Cells of Chlorella protothecoides1 ) TETSUAKI OSAFUNE and EIJI HASE The Institute of Applied Microbiology, the University of Tokyo, Japan Key T er mIn d e x: chloroplast, fine structure, degeneration, chlorophyll metabolism, glucosebleaching; Chlorella protothecoides

Summary When green cells of Chlorella protothecoides were incubated, in the dark, in a medium containing glucose but no nitrogen source, many starch-like granules were observed filling the spaces delimited by the lamellae in the swollen chloroplast. The chloroplast .la.mellae were then broken into shorter pieces, which gradually disappeared concomitantly with the progress of cell bleaching (chlorophyll degradation). In the meantime, the chloroplast envelope produced protuberances into the cytoplasm, and the the starch-like granules seemed to move into the protuberances which were subsequently detached from the chloroplast body. In the strongly bleached cell("glucose-bleached" cell) there was a small plastid containing apparently no internal structures. When the "glucose-bleached" cells were incubated, in the light, in a medium containing a nitrogen source but no organic carbon source, an active synthesis of chlorophyll proceeded after a lag time. The structures of developing plastids in the cells before and immediately after the lag time of chlorophyll formation were examined. The shape of the degenerate plastid was like an irregularly deformed vase, and subsequently changed into an amoeboid form extending pseudopod-like arms into the cytoplasm. The extended arms of the plastid were in close association with cytoplasmic vesicles, suggesting that certain cytoplasmic substance(s) may be transferred to the developing plastid through the structural association.

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Introduction

Previous studies (SHIHIRA-IsHIKAWA and HASE 1964, OH-HAMA et al. 1965, HASE 1971) have demonstrated that profound degeneration and regeneration of chloroplasts are induced in Chlorella protothecoides cells by the control of nutritional as well as light conditions. When green cells are incubated in a medium containing a high concentration of glucose or other metabolizable organic carbon compounds but no nitrogen source, they are strongly bleached, either in the light or in darkness, becoming "glucose-bleached" cells containing degenerate plastids (Fig. 1). When the glucose-bleached cells are incubated in a medium containing a nitrogen source but no organic carbon source, they are turned into normal green cells in the light, while in the dark they become pale green c.ells ("etiolated" cells) containing only partially organized plastids. This report describes some structural characteristics of degenerating chloroplasts in the "glucose-bleaching" cells and of developing plastids in the re-greening cells in the 1) Dedicated to Prof. KURT MOTHES on the occasion of his 75th birthday.

T. OSAFUNE and E. HASE

534

ETIOLATED CELL

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BLEACHED CELL ( GEGENRATED ) PLASTID

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Fig. 1. A diagrammatic representation of the processes of de- and re-generation of chloroplast in Chlorella protothecoides.

light. The results were discussed in correlation with our previous results of biochemical and physiological studies. Material and Methods Chlorella protothecoides was obtained from the Algal Culture Collection of the Institute of Applied Microbiology, the University of Tokyol). Bleaching experiment. - The starting green cells were obtained by growing the alga in a medium containing urea (initial conc.: 0.5 %), glucose (initial conc.: 0.1 %) and the basal nutrients2 ) under illumination (light intensity: 2000 lux) for 5 days at 25°C. These cells were incubated, in the dark, in a medium containing 1 % glucose and the basal nutrients. The algal suspension was placed in a MONOD-type vessel and shaken at 25°C. The chlorophyll contents of the bleaching cells are shown in Table 1.

Table 1. Chlorophyll contents of the bleaching cells (p.moles per ml packed cell volume) Incubation time/hour)

o

6

12

4.4

2.8

2.5

24

1.5

Chlorophyll was determined with the methanol extract by the method of OGAWA and SHIBATA (1965). Greening experiment. - The green cells obtained as above were incubated in a medium containing glucose (initial conc.: 1 %), urea (initial conc.: 0.1 %) and the basal nutrients, and shaken for 5 days at 25°C. The cells thus obtained contained no chlorophyll and used as the starting "glucosebleached" cells in the greening experiment. These cells were transferred to a flat oblong culture vessel (cf. HASE and l\fORIMl:RA 1971) containing 0.1 % NH4 CI as nitrogen source and the basal nutrients. The culture was illuminated with daylight fluorescent lamps (the light intensity at the surface of the vessel: 2000 lux) and aerated with air at 25°C. Under these conditions an active chlorophyll formation started after the lag time of about 30 hours. 1) This strain was supplied originally from the algal culture collection at University of Indiana, U.S.A., being denoted as strain AC No. 25. 2) Per liter: KH2 P04 0.7 g, K 2 HP04 0.3 g, )IgS0 4 • 7H2 0 0.3 g, FeS0 4 • 7 H 2 0 2.8 mg, thiamine hydrochloride 10 p.g, ARNON'S "As" solution (ARNON 1938) 1 ml; pH 6.2.

Chloroplast Structure During "Glucose-bleaching" and Regeneration

535

Electron microscopy. - Glutaraldehyde was added to a cell suspension in culture medium immediately after harvesting to give a final concentration of 5 %. The cell suspension was left standing in an iced water-bath for 1-1.5 hours and centrifuged at 1500 x g. The sedimented cells were washed three times with the basal nutrient solution containing none of FeS04 , ARNON'S "Aa" microelements and thiamine hydrochloride. The sample was then suspended in 1 % KMn0 4 solution for 4 hours at room temperature. After washing two times with deionized water, the sample was stained with 0.5 % uranyl aetate solution and embedded in 2 % agar, which was cut into 1 mmS blocks. The agar blocks were dehydrated successively with an ethanol series (30 - 90 %) and an acetone series (90-100~~), then were embedded in Epon 812 (LUFT 1961), unless otherwise mentioned. Thin sections were cut on a PORTER-BLUM MT-1 with a glass knife. The sections were stained with lead salt solution (SATO 1968), after which they were examined with a JEM-7 A electron microscope.

Results and Discussion

Fig. 2 -(1) reproduces a section of a green cell in which the chloroplast lamellae show a normal arrangement with stacking of several thylakoids and a few starch granules exist between the lamellae. An initial structural change in the chloroplast after the incubation of the green cells with glucose was an accumulation of many starch-like granules among the lamellae as observed previously (SHIHIRA-IsHIKAWA and RASE 1964) [see Fig. 2-(2)]. This is in accordance with the previous result that added glucose was rapidly incorporated into polyglucose during the bleaching of green cells (MATSUKA et al. 1969, MATSUKA and RASE 1966). As seen from Fig. 3-(3), the chloroplast lamellae were then cut into shorter pieces, which disappeared later concomitantly with the progress of cell bleaching or chlorophyll degradation (OSHIO and RASE 1969 a, 1969b). In the meantime the chloroplast envelope produced protuberances into the cytoplasm, and the starch-like granules seemed to move into the protuberances, as seen from Figs. 3-(3), (4) and (5). These protuberances were then detached from the chloroplast and were accumulated in the cytoplasm as vesicles, as may be seen from Fig. 3-(4). These vesicles eventually disappeared, and in the glucose bleached cell there was a small degenerate plastid in a narrow space of the protoplasm surrounded by large vacuoles [cf. Fig. 4-(6)]. Although the starch-like granules were not recognized as such in the glucose-bleached cells, it is to be noted that the content of "insoluble carbohydrate" (MATSUKA et al. 1966) or the activity for incorporating added glucose into polyglucose (MATSUKA et al. 1969) remained high in the bleached cells. Previous studies revealed that when the green cells were incubated in the glucose medium, the activity of the cells for assimilating glucose into lipids was markedly increased (MATSUKA et al. 1969). It was suggested that the formation of large amounts of lipids probably is causally related to the induction of cell bleaching (MATSUKA and RASE 1969). The present results shows that it may also be correlated with the degradation of the chloroplast structures. When the glucose-bleached cells are incubated, in the light, in a medium containing nitrogen source but no organic carbon source, an active chlorophyll formation starts after a lag time of 15-30 hours. In the experiment described below, the lag time was about 30 hours. Fig. 4-(6) shows a section of a cell taken from the culture at the 6th hour from the beginning of incubation in a nitrogen medium. Apparently this figure is similar to that obtained with the glucose-bleached cell. Fig. 4-(7) represents a plastid

536

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Chloroplast Structure During "Glucose-bleaching" and Regeneration

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537

at a more advanced stage. The shape of the plastids at such early stages of development was like an irregularly deformed vase, and the section in Fig. 4-(7) may represent a profile of the vase cut near the upper edge in parallel with the plane of its bottom, the two layers of membranes in the figure being the outer and the inner surfaces of the vase. In this figure discs are seen existing in a narrow space between the two surfaces. At further advanced· stages of development, the chloroplast showed an amoeboid form extending pseudopod-like arms, as seen from Figs. 4-(8), 5-(9), (10) and (12). The "pseudopods" of the developing chloroplasts were in close association with vesicles in the cytoplasm. In Fig. 4-(8) a "pseudopod" containing discs is extended from a developing chloroplast and is embracing a cytoplasmic vesicle. Figs. 5-(9) and (10) show another example of the association of the chloroplast "pseudopods" with a cytoplasmic vesicle. As seen from Fig. 5 -(12), the developing chloroplasts were frequently observed to extend long and short "pseudopods" in various directions in the cytoplasm and to be in association with several cytoplasmic vesicles simultaneous. Such a behaviour of the developing chloroplasts may facilitate the uptake of certain substances of cytoplasmic origin for the construction of chloroplast components. It is to be noted that chloroplast thylakoids seemed to develop in the region of the chloroplast stroma in close proximity to the embranced cytoplasmic vesicles, as may be seen from these figures, especially Fig. 5-(11). Previous investigations have shown that the syntheses of certain chloroplast substances depend on the cytoplasm (cf. RASE 1971, OSHIO and RASE 1972, OCHIAI-YANAGI et al. 1973, OH-HAMA and RASE 1975). Recently it was found that the synthesis of main chloroplast lamellar proteins in the greening cells of Chlorella protothecoides were strongly inhibited by cycloheximide, suggesting that these proteins are supplied from the cytoplasm (RAYAKAWA, OH-HAMA and RASE, unpublished). The real mechanism of transfer of these cytoplasmic substances to the developing chloroplasts is a problem to be elucidated in the subsequent work. Acknowledgements We are grateful to Professor HIROSHI TAMIYA for hi~ encouragement in this work, and to Messrs. S. HAYAKAWA and T. AOKI for their help in the experiments and in the preparation of this manuscript. This work was aided by grants from the Ministry of Education.

Fig. 2. (1) Section of a green cell. CP: chloroplast, N: nucleus, M: mitochondrion, V: vacuole. The section was obtained as described in Methods, except that the cell sample was embedded in Vestopal W (OSAFUNE et al. 1972, KUSHID_~ and FUJITA 1968).

(2) Section of a bleaching cell. The cell sample was taken from the culture at the 6th hour of incubation with glucose. S: starchlike granule. Note many starch-like granules accumulated among the lamellae (L) in a swollen chloroplast.

538

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Chloroplast Structure During "Glucose-bleaching" and Regeneration

541

Fig. 3. (3) Section of a bleaching cell. The cell sample was taken from the culture at the 12th hour of incubation with glucose. Note shorter pieces of the chloroplast lamellae and a protuberance of the chloroplast envelope indicated by the arrow. (4 and 5). Two sections of a bleaching cell from the same culture as that in No.3. Note a characteristic protuberance of the chloroplast envelope in which the starch-like granules are contained. The arrows indicate a possible site of detachment of the protuberance from the main chloroplast body. Note also several vesicles existing in proximity to the protuberance which seemed to be the detached fragments of chloroplast. Fig. 4. (6) Section of a cell from the culture obtained 6 hours after the incubation of glucose-bleached cells in a glucose-free and nitrogen source-containing medium in the 11:ght (greening experiment). The arrow shows a still degenerate plastid.

(7) Section of a developing plastid. The cell sample was obtained from the 12th hour culture of greening experiment. D: discs. (8) Section of a developing chloroplast Irith an extended arm embracing a cytoplasmic vesicle. Note discs (D) in the extended arm and apparently accomplished lamellae in the main chloroplast body. The cell sample was taken from the 30th hour c!llture of greening experiment.

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Fig. 5. (9 and 10) A mode of embracing a cytoplasmic vesicle with extended arms of the developing chloroplast. The cell sample was obtained from the 30th hour culture of greening expermient. The figure in No. 10 was obtained by tilting the section in No.9 at 30°. Note the membrane of the cytoplasmic vesicle lying along the chloroplast envelope. (11) Section of a cytoplasmic vesicle surrounded by the developing chloroplast. Note developing thylakoids (indicated by the arrow) in the region of the chloroplast stroma in close proximity to the vesicle. The cell sample was taken from the 30th hour culture of greening experiment. (12) Section of a developing chloroplast. The cell sample was obtained from the 30th hour culture of greening experiment. Note the amoeboid form of the chloroplast with a long extended "pseudopod" in association with cytoplasmic vesicles, and the membranes surrounding a vesicle in the central part of the chloroplast.

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References

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ARNON, D. 1., Microelements in culture-solution experiments with higher plants. Amer. J. Bot. 21) 322-325 (1938). HASE, E., Studies on the metabolism of nucleic acid and protein associated with the processes of deand re-generation of chloroplasts in Chlorella protothecoides. In: Autonomy and Biogenesis of Mitochondria and Chloroplasts. North-Holland Publishing Co., Amsterdam, pp. 434-446 (1971). and l\!ORIMURA, Y., Synchronous culture of Chlorella. In: Methods in Enzymology XXIII, Photosynthesis Part A. Academic Press, New York 1971, pp. 78-84. KUSHIDA, H., and FUJITA, K., Methyl methacrylate as an auxiliary to infiltration for embedding with Vestopal W. J. Electron Microscopy 17, 349-350 (1968). LUFT, J. H., Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414 (1961).

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T. OSAFUNE and E. RASE, Chloroplast Structure During "Glucose-bleaching" etc,

MATSUKA, .1.1., OTSUKA, R., and RASE, E., Changes in contents of carbohydrate and fatty acid in the cells of Chlorella protothecoides during the processes of de- and re-generation of chloroplasts. Plant & Cell Physiol. 7, 651-662 (1966). and RASE, E., Effect of cycloheximide on the process of "glucose-bleaching" in Chlorella protothecoides. Plant & Cell Physiol. 10, 277 - 282 (1969). MIYACHI, S., and RASE, E., Further studies on the metabolism of glucose in the process of "glucose-bleaching" of Chlorella protothecoides. Plant & Cell Physiol. 10, 513-526 (1969). OCHIAI-YANAGI, S., and RASE, E., Studies on chlorophyll formation in Chlorella protothecoides II. Enhancing effects of light and suppressive effects of glucose on the development of porphyrinsynthesizing activity during light-induced greening of etiolated algal cells. Plant & Cell Physiol. 13,747-762 (1972). MATSL"KA, }I., and RASE, E., Studies on chlorophyll formation in Chlorella protothecoides III. Effects of chloramphenicol, cycloheximide, and ethionine on chlorophyll formation. Plant & Cell Physiol. 14, 299-305.(1973). OGAWA, T., and SHIBATA, K., A sensitive method for determining chlorophyll b in plant extracts. Photochem. Photobiol. 4, 193 - 200 (1965). OH-HAMA, T., SHIHIARA-IsHIKAWA, I., and RASE, E., Development of photosynthetic activities during the process of chloroplast formation in Chlorella protothecoides. Plant & Cell Physiol. 6, 743-760 (1965). and RASE, E., Syntheses of o-aminolevulinic acid and chlorophyll during the process of chloroplast formation in Chlorella protothecoides. Plant & Cell Physiol. 16, 297 -303 (1975). OSAFUNE, T., MIHARA, S., RASE, E., and OHKURO, I., Electron microscope studies of the vegetative cellular life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture II. Association of mitochondria and the chloroplast at an early developmental stage. Plant & Cell Physiol. 13, 981- 989 (1972). OSHIO, Y., and RASE, E., Studies on red pigments excreted by cells of Chlorella protothecoides during the process of bleaching induced by glucose or acetate. I. Chemical properties of the red pigments. Plant & Cell Physiol. 10, 41-49 (1969a). - Studies on red pigments excreted by cells of Chlorella protothecoides during the process of bleaching induced by glucose or acetate II. Mode of formation of the red pigments. Plant & Cell Physiol. 10, 51-59 (1969b). - Changes of ribulose 1,5-diphosphate carboxylase level during the processes .of degeneration and regeneration of chloroplasts in Chlorella protothecoides. Plant & Cell Physiol. 13, 955-963 (1972). SATO, T., A modified method for lead staining of thin sections. J. Electron Microscopy 17, 158-159 (1968). SHIHIRA-IsHIKAWA I., and RASE, E., Nutritional control of cell pigmentation in Chlorella protothecoides with special reference to the degeneration of chloroplast induced by glucose. Plant & Cell Physiol. 0, 227 - 240 (1964). Received March 17,1975. Author's present address: T. OSAFUNE, Department of Microbiology, Tokyo Medical College, Tokyo 160 and E. RASE, Institute of Applied Microbiology, the University of Tokyo, Tokyo 113. (Japan).