Entwicklung und Struktur der Proplastiden

In this study the proplastid development in embryonic cells is described for the apical meristem of Elodea canadensis, embryo sacs from Lilies, and Begonia leaf buds. The formation of these cell organelles originates with submicroscopical particles which consist of a homogeneous stroma with a surrounding double membrane. When these proplastids reach an average size of 1 µ, the inner layer of the membrane begins to invaginate into the stroma. This process is comparable to tubuli formation in mitochondria. Under growth conditions with sufficient exposure to light, the development of the grana and stroma lamellae proceeds without interruption. If the plants are kept in the dark, small vesicles are formed which accumulate in the prolamellar body of the proplastids. After illumination these elementary vesicles merge to form membranes which evolve into grana and stroma lamellae. The structural similarity of the early proplastid stages with the mitochondria seems to indicate that there exists some phylogenetic relationship between the two cell organelles.     

THE ULTRASTRUCTURAL MORPHOLOGY AND ONTOGENY OF OAT COLEOPTILE PLASTIDS

Structurally similar proplastids occur in the shoot, scutellum, and root of the oat embryo at the start of germination. These proplastids follow several pathways of differentiation, depending on their location within an organ and on previous exposure to light. During the first 24 hr of germination morphologically similar amyloplasts are formed from the preexisting proplastids in most of the cells of the seedling. After about 24 hr in the light, unique chloroplasts begin to develop in a subepidermal ring of small cortical parenchyma cells in the coleoptile and give the organ a pale green color. At 48 and 72 hr the coleoptile chloroplasts and etioplasts are conspicuously different from the corresponding leaf plastids in morphology and ontogeny but contain typical photosynthetic grana and prolamellar bodies. Study of the ontogeny of plastids in the epidermal and nongreening parenchymal regions of dark grown coleoptiles shows that these plastids undergo significant losses in starch content, and some increase of membranes within the plastid, related to the age of the cell. Light has little effect on the structure of these plastids. It is suggested that the ontogeny of all the plastid types of the oat seedling begins with a common precursor—a relatively simple proplastid that is present at the time of germination. Starch grains showing two distinct types of erosion, apparently enzymatic, were observed in oat coleoptile plastids. In one type (grooved appearance) the starch grains are consistently associated with plastid membranes, while in the other type (irregular, spiny appearance) the starch grains are associated with the plastid stroma only. We suggest that there are two enzyme systems for metabolizing starch in oat plastids—one membrane-bound and the other free in the stroma.     

AN ULTRASTRUCTURAL STUDY OF CHLOROPLAST STRUCTURE AND DEDIFFERENTIATION IN TISSUE CULTURES OF STREPTANTHUS TORTUOSUS (CRUCIFERAE)

Cells of Streptanthus tortuosus callus tissue contain chloroplasts when cultured in a liquid medium in the light. Similar cells grown in the dark contain proplastids that fail to develop prolamellar bodies but do contain a complex of loosely-associated membranes. When green, light-grown cultures are cut into small pieces and subcultured to a fresh culture medium, they become bleached even though maintained under the same illumination. The fine structure of the chloroplasts and the chlorophyll content of the cells indicate a dedifferentiation of the chloroplasts to a proplastid state during the early culture period. The changes in the ultrastructure of the plastids are paralleled by a dedifferentiation of the vacuolate cells to a less differentiated, meristematic state. Subsequent growth in the light results in a re-formation of chloroplasts and an increase in the chlorophyll content of the cells. The period of chloroplast redevelopment is associated with the re-formation of large central vacuoles in the cultured cells. Invaginations of the inner membrane of the plastid envelope occur at all stages of plastid development and are not lost during the period of grana degeneration. The proplastids formed from the dedifferentiation of the chloroplasts contain a large number of these invaginations and the redevelopment of grana is associated with a change in the electron density of the invaginating membranes. The degradation of the chlorophyll-containing membranes of the grana occurs during a period of rapid cytoplasmic synthesis induced by the fresh supply of nutrients in the culture medium. These results suggest that the high levels of nutrients may act directly on the chloroplasts and cause their dedifferentiation or that the rapid cell growth induced by the nutrients may cause a degradation of the membrane proteins in the grana of the chloroplasts and an incorporation of the released amino acids into non-plastid components of the cytoplasm.     

CHANGES INDUCED BY LOW LIGHT INTENSITIES ON THE PROLAMELLAR BODY OF 8-DAY,DARK-GROWN SEEDLINGS

Etioplasts of 8-day-old, dark-grown seedlings of Phaseolus vulgaris contain large, crystalline prolamellar bodies. The basic structural unit within the prolamellar body is a six-pointed star (star module) with four tubules fusing at each of the nodes. With sufficient illumination some of the tubules are withdrawn and the crystalline prolamellar body transforms to a complex tangle of tubules, the reacted prolamellar body. In vivo spectrophotometry and electron microscopic observations were carried out on portions of the same leaves after varying periods of illumination with low light intensity. Protochlorophyllide transformation was normal. However, the structural changes are not closely tied to protochlorophyllide conversion. The pigment conversion is complete after 20 sec of illumination, but 80% of the prolamellar bodies are still in the crystalline form after 20 min of illumination. After 1 and 2 hr of illumination all prolamellar bodies are reacted. After 4 hr of continuous illumination 35%, and by 12 hr 60%, of the prolamellar bodies returned to the crystalline form. Spectrophotometric evidence and presence of grana show chlorophyll synthesis during this period. The coexistence of grana and the crystalline prolamellar body indicates that when insufficient photosynthetic membrane constituents are provided by the photo-reactions, under low light intensity, the membranes of the reacted prolamellar body will be forced to reform a crystalline prolamellar body.     

Ultrastructural Development of Chloroplasts in Sacred Lotus Embryo Bud under Invisible Light

The present paper reports that the development ultrastructural observations of chloroplasts from sacred lotus (Nelumbo nucifera) embryo buds under invisible light. Embryo bud of sacred lotus is enclosed by three layers of thick integument (pericap, seed coat and thick fleshy cotyledons). During the period of the formation of embryo bud, it remained in dark condition, but turned from pale yellow to bluish-green. It was noteworthy that chloroplasts of the embryo bud had well developed giant grana under invisible light. Their developmental pathway in sacred lotus, however, was different from those of other higher plants grown under sunlight, intermittent light, or even in dark conditions (Fig. 1). The chloroplast development of embryo buds in Sacred lotus seeds in invisible light underwent only in the following three stages: (1) In the first stage the development was similar to that from other higher plants, the inner envelope membranes of the proplastids were invaginating. (2) In the second stage, a proplastid centre composed of prolamellar bodies (PLB)with semicrystalline structure was formed, and was accompanied by one or two huge starch grains in almost each proplastid. In the meantime, prothylakoid membranes extended parallelly from the plastid centre in three forms: (a) One plastid centre extending parallelly prothylakoid membranes from itself in one direction; (b) The same to (a), but extending in two directions; (c) Two plastid centres extending parallelly prothylakoid membranes between the centres. (3) In the third stage, grana and stroma thylakoid membranes of chloroplasts were formed. It is to be noted that most of chloroplasts had only one or two giant grana which often extended across the entire chloroplast body, and the length of the grana thylakoid membranes of the chloroplasts from embryo bud in Sacred lotus is 3 to 5 times as many as that in other higher plants. However, their stromatic thylakoid membranes were rather rare and very short. The giant grana were squeezed to the margin of the chloroplast envelope by one or two huge starch grains.     

FINE STRUCTURAL CHANGES IN PROPLASTIDS DURING PHOTODESTRUCTION OF PIGMENTS

Etiolated bean leaves supplied δ-amino-levulinic acid in the dark synthesize large amounts of protochlorophyllide which is not converted to chlorophyllide upon illumination of the leaves. The fine structure of the proplastids is not affected by the treatment. When leaves containing "inactive" protochlorophyllide are exposed to light of 700 ft-c for 3 hours, they lose practically all their green pigments. During this period large stacks of closed membrane structures are built up in the region of the prolamellar body. These lamellar structures remain even when no or only traces of pigment are left in the leaves. In untreated control leaves the pigment content remained constant during similar illumination and the structural changes in the plastids consisted of a rearrangement of the vesicles from the prolamellar bodies into strands dispersed through the stroma; lamellae and grana formation occurred later.     

Separation and Ultrastructure of Proplastids from Dark-grown Euglena Cells

A procedure for the separation of proplastids free of mitochondria from dark-grown Euglena cells has been developed. A fraction enriched in proplastids was used for freeze-etching study of proplastid structure. The prolamellar body in freeze-etched replicas appeared sponge-like, with thylakoids, often vesicular, emerging from it. The prolamellar body and the thylakoids were covered by particles of about 100Å in diameter. No larger particles, typical of light-grown chloroplasts, were observed.     

The mitotic apparatus. Identification of the major soluble component of the glycol-isolated mitotic apparatus

Particles having ribosome-like characteristics are described in proplastids of dark-grown wheat seedlings as the membranes of the prolamellar body become transformed, under the influence of light, into grana and fret membranes. Three arrangements of particles were noted: (1) a random distribution of discrete particles; (2) particles occurring in helices or parallel rows; and (3) particles arranged in rough squares with six to eight particles per side. It is possible that the third type of particle is a cross-section of long parallel rods. A particle ranges in size from 170 to 220 A, those of group three being somewhat smaller. The particulates vary from diamond shaped with smooth surfaces to circular with irregular surfaces. These particles have the characteristics of ribosomes as visualized by the electron microscope: they are preserved by glutaraldehyde and osmium tetroxide, they stain intensely with uranyl acetate, and are digested by RNase. Their properties do not coincide with those of viruses, smog-induced particles, stromacenter particles, or phytoferritin. They are frequently adjacent to membranes but never attached to membranes. The involvement of ribosomes in membrane development is discussed.     

The ultrastructural development of plastids in leaves of maize plants exposed to continuous illumination

Summary Chloroplast differentiation in relation to increasing leaf age has been investigated in maize plants exposed to continuous illumination. In the young leaves the proplastids differentiate into chloroplasts containing well organized grana as well as prolamellar bodies. In the older leaves, while plastids differentiate, the prolamellar bodies are no longer detectable. Chloroplast ability to build up prolamellar bodies does not seems so much a light dependent process as it is affected by cell differentiation rate.Supported by a grant of C.N.R.     

CHLOROPLAST DEVELOPMENT AND ULTRESTRUCTURE IN THE FRESHWATER RED ALGA BATRACHOSPERMUM1

Chloroplast development and ultrastructure of the freshwater red alga Batrachospermum moniliforme are described. Chloroplasts develop from proplastids which have a double-membraned chloroplast envelope and a parallel double-membraned outer photo-synthetic lamella. Of these 2 double-membraned structures of the proplastid, only the outermost pho-tosynthetic lamella functions in production of further lamellae. The mature chloroplast consists of 2 or more concentric lamellae and a variable number of nonconcentric lamellae. These lamellae are not dense, uninterrupted sheets as described for other red algae, but are largely constructed of tubules, lying side by side, that form interrupted lamellar sheets. The possible physiological significance of lamellar interruptions in providing path-ways for movement of materials in the chloroplast stroma is discussed.     

FINE STRUCTURE AND PIGMENT CONVERSION IN ISOLATED ETIOLATED PROPLASTIDS

Proplastids containing a prolamellar body were isolated from leaves of etiolated bean plants. The isolation methods do not necessarily lead to destruction of their submicroscopic structure and most of the isolated proplastids show well preserved outer membranes, lamellar strands, and the prolamellar body. Morphological intactness of the proplastids varies; certain leaf fractions contain single prolamellar bodies as well as proplastids. Since pellets after centrifugation between 350 g and 1000 to 3000 g contain intact proplastids and, as was shown by quantitative experiments, the same fractions show photoconversion of protochlorophyll to chlorophyll, it is supposed that the isolated particles probably retain many of the properties which are characteristic of them in situ. Isolated proplastids may thus be a valuable tool in investigations on the development of the photosynthetic apparatus.     

FORMATION OF THE PROLAMELLAR BODY IN 8-DAY,DARK-GROWN SEEDLINGS

The development of the prolamellar body in etioplasts of dark-grown seedlings of Phaseolus vulgaris is followed through the 8th day. From 2 to 6 days there is an increase in plastid size and starch content and synthesis of a system of porous lamellae which appear to arise, as such, from the inner component of the plastid envelope. From 6 to 8 days much of the starch disappears accompanied by rapid membrane synthesis resulting in an extensive prolamellar body. A model of the prolamellar body is discussed in which the basic structural unit is a six-pointed star with four tubules joining at each node. Observation of face views of the porous peripheral lamellae at their juncture with the prolamellar body suggests the origin of the prolamellar body by the continued contraction of the porous lamellae and the formation of interconnecting tubules between adjacent lamellae. The pores of the peripheral lamellae appear to correspond to the areas of stroma within each star module. Short lengths of membranes of individual peripheral lamellae fuse, forming short overlaps which resemble small, two-compartmented grana. It is postulated that this is the initial step in grana formation.     

Plastid development in seedlings of Echinochloa crus-galli var. oryzicola under anoxic germination conditions

Plastid development in the primary leaf of Echinochloa crus-galli (L.) Beauv. var. oryzicola (Vasing.) Ohwi was followed during 5 d of anoxic germination and growth. Plastids develop slowly from simple spheroidal proplastids into larger pleomorphic plastids with several stromal membranes and many peripheral membrane vesicles. A small prolamellar body is present at 96 h with perforated (pro)thylakoids extending into the stroma. Changes in starch grains and plastoglobuli are evidence of carbohydrate and lipid metabolism. Plastid division is indicated by dumbbell plastid profiles after 4 d of anoxia. These results demonstrate that plastids not only maintain their integrity during anaerobic germination but also show developmental changes involving an increase in internal membrane complexity, although to a lesser extent than in etiolated shoots.Abbreviation PLB prolamellar body Scientific paper No. 6167. College of Agriculture, Washington State University, Pullman     

Ultrastructural changes in isolated plastids

Summary Etioplasts were isolated from maize leaves and the changes in their ultrastructure were followed in light and in darkness for several hours. It has been shown that the regular crystalline structures of prolamellar bodies, present after the isolation in darkness, disappear after 30 to 60 minutes of illumination, and long straight tubules appear within prolamellar bodies. Their appearance is influenced by the molarity of the isolation medium used, by light intensity, duration of illumination and by the temperature at which the isolates are kept. Long tubules appear, however, also in isolated etioplasts incubated for several hours in complete darkness.In isolates illuminated for 2–3 hours long tubules disappear again, and prolamellar bodies produced eventually consist of irregularly connected short tubules. In prolamellar bodies, regions with regular and very dense arrangement of tubules sometimes develop at this stage. The thylakoids (usually perforated) are now arranged concentrically in the plastids. True grana or poly-thylakoids can never be found in isolated etioplasts, not even when the etioplasts have been illuminated for 6 hours or more (up to 24).The present investigations have indicated that in isolated etioplasts in light, tubular elements, which build up the prolamellar bodies, cannot normally be transformed into thylakoids as is the case with intact tissue.The survival of isolated etioplasts is limited at present, and for this reason changes in their fine structure could be followed successfully for as long as 6 hours (in light at 15 °C), although a certain percentage of plastids survive up to 24 hours.     

Ultrastructural Study of Proplastid Ontogeny in Tobacco Mesophyll Cells in Vitro

Since the discovery of plastid DNA the continuity of plastids has well been established. It is known that in plant cultures a form of plastid can differentiate into others. However, only a little has been made in studing chloroplast dedifferentiation in vitro. In the work present here, we reported on ultrastructural changes of chloroplasts dedifferentiation and the proplastid origin in the mesophyll cells of cultured tobacco leaf explant. Fully expanded leaves of haploid tobacco (cv. Ge Xin No. 1) were cut into pieces of 5–6 mm width. These were inoculated on MS medium supplemented with 1 mg/L 2,4-D and 1 mg/l kinetin. The cultures were maintained at (30±2) ℃ and illuminatied by a bank of fluorescent lamps. For electronmicroseopic investigation, after 0, 1, 2, 3, 6 days of culture small leaf fragments were cut off along the cut edges of the explants. The samples were fixed and processed in the manner as described earlier. The sections were examined with a Hitachi HU-11A or a JEM-100CX electronmicroscope. Electronmicroscopic observation shows that the uncultured mesophyll cells are highly vacuolete, with a thin peripheral layer of cytoplasm in which a nucleus and some chloroplasts and other organelles are found in it. But these cells do not contain proplastids (Fig. l). In the explants cultured for 1 day there are no obviously changes in mesophyll cells, except a few cytoplasmic strands extend from periphery to central vacuole. At 2 days of culture quite obvious changes can be detected. A increase in the amount of cytoplasm becomes apparent and transvacuolar cytoplasmic strands grow up. Following cytoplasmic growth, the nucleus and chloroplasts move away from the peripheral cytoplasm and enter the central vacuolate zone (Fig. 2). At this stage some of mesophyll cells have completed the first cell division. After 3 days of culture numerous mesophyll cells have undergone several divisions and formed multicellular masses. In those subdivided cells a more important change of the chloroplasts is the occurrence of protrusions which we call proplastid buds. This phenomenon has also been named as chloroplast budding. According to observations on a large amount of sections chloroplast budding is a common phenomenon in the dedifferentiating mesophyll cells of tobacco leaf explants. Fig ure 3 exhibits a typical profile of a chloroplast with a proplastid bud. The proplastid buds observed are generally long-oval in shape and 1.0–2.5 μm long and about 0.5–0.7 μm thick. These dimensions agree with those of proplastids in meristematie cells. Inside of proplastids ribosomes and electron opaque areas containing DNA fibrils can be seen (Fig. 3). Near the proplastid buds proplastids can often be found (Fig.5). According to above observations we can conclude that the proplastids in dedifferentiating mesophyll cells originate from the proplastid buds by chloroplast budding. The newly formed proplastids usually surround the nucleus and sometimes undergo equal division to increase their number (Figs.5, 6). There are no inner membranes in the newly formed proplastids except vesicles connected with inner membrane of the envelope (Fig.7). While the proplastids are continuously produced, the chloroplasts themselves are filled with starch and gradually turned to large amyloplasts (Fig.5). On the other hand, a few of chloroplasts can divide into equal parts following the chloroplast budding (Fig.4). Israel and Steward (1967) suggested that when cultured carrot cells developed into plantlets the chloroplasts turned into leucoplastids, chromoplastids or proplastids. However, they did not describe how chloroplast became a proplastid. Several investigators reported that the chloroplasts in the dedifferentiating cells gradually lost their grana and intergranal lamellae and then became eueoplasts or proplastids. But according to our observation in tobacco explants, the initiation of proplastids is due to unequal division of chloroplasts, i.e. “budding fission” as described by Malzan and Miihlethaler in Splachnum ampullaceum. Since the proplastid is an organelle characteristic of meristematie cells, the ontogeny of proplastids and its control mechanism should be very important in studing cell dedifferentiation.     

Ultrastructure of Protein Bodies and Plastids in Cotyledon of Nelumbo nucifera Gaertn.at Seed Germination

The present article deals mainly with the formation and dissolution of protein bodies and development of plastids in cotyledon cells of Nelumbo nucifera during seed germination. Electron microscopic studies reveal that protein bodies are formed after imbibition of the cotyledons before germination. They are produced through accumulation of protein material in small vacuoles delivered from the exudates of endoplasmic reticulum or by fragmentation of endoplasmic reticulum itself. In the period of germination, most of the material in the protein bodies dissolute and they coalesce with each other forming large vacuoles. The protein residue of the vacuoles condenses into small blocks with high electron density adhering to the tonoplast or freely floating in the vacuole. Thus, it suggests that the protein bodies of the germinating N. nucifera cotyledons are originated from vacuoles formed by endoplasmic reticulum. Part of the plastids found in cotyledonous cells of mature N. nucifera seeds exists as proplastids. They develop continuously after imbibition of the cotyledons. During the period of seed germination, many concentric lamellae are developed along the plastid membrane on which they later coalesce with the neighboring concentric lameUae forming loosely organized prolamellar bodies which condense into paracrystalline lattices. No ribosomes are present in the inter spaces of paracrystatline lattice. One to several prolamellar bodies can be developed in one plastid.     

Plastids in the Quiescent Embryo and Young Seedlings of the Chloroembryophyte Citrus nobiisxCitrus aurantium amara pumila

Plastid ultrastructure has been investigated in green clementineembryos. Research has been extended to both dark- and light-grownseedlings in order to study the fate of the chioroplasts showinglarge membrane stacks which had been found in the more differentiatedembryo tissues. During germination in the dark the chloroplastslose their membrane stacks and prolamellar bodies are visible.In the light their thylakoid system grows more abundant andis well arranged. Therefore in both cases the embryo chloroplastsdevelop internal organization similar to that of the plastidsdifferentiated from the proplastids of the embryo meristems.The possible significance and function of the embryo chloroplastsis discussed. Citrus nobilis x C, auranhium amara pumila, clementine, embryo, plastid, ultrastructure     

ULTRASTRUCTURE OF CHLOROPLASTS AND CHROMOPLASTS IN CAPSICUM ANNUUM I. THYLAKOID MEMBRANE CHANGES DURING FRUIT RIPENING

Developing chromoplasts in the fruit of Capsicum annuum were examined by electron microscopy. Special attention was given to changes in the thylakoid system. All grana and some intergranal thylakoids in the mature chromoplasts of the seven cultivars studied underwent lysis. The particulate nature of the granal membranes disappeared during lysis before the relationship between the partitions and locules was obscured. The changes during lysis support the globular concept of membrane structure. The selective lysis of the synaptic membranes of the granal partitions may be attributed to their distinctive composition and structure. Lipid globules (osmio-philic) did not accumulate in the immediate region of granal lysis, indicating that they are not directly derived from membranes undergoing degradation. During and following granal lysis a profuse development of intergranal thylakoid membranes occurred in several cultivars. In some instances a thylakoid plexus (prolamellar body) was formed. This specialized structure of the thylakoid system occurs in the chromoplasts of other species as well as in other types of plastids. Extensive, concentrically arranged thylakoid sheets with specific interspaced membrane relationships were frequently associated with the plexus. Several types of membrane associations and interrelationships in the plastid are described. An analysis of one type of membrane configuration, the thylakoid sheets, indicated that one method of growth may be through intussusception into the original membrane. The development of thylakoid plexes and of extensive thylakoid sheets during or after granal lysis indicates that dynamic synthetic activities occur in the chromoplasts of some cultivars of pepper during fruit ripening.     

Stimulation of Plastidogenesis Induced by 5-Azacytidine in Euglena gracilis Klebs

When etiolated Euglena gracilis was treated with 10 mM 5-azacytidine (5-azaC), an inhibitor of DNA methylation, stimulation of plastidogenesis in both dark and light conditions was observed. The phenomenon occurred in 10–15% of the cells possibly due to the asynchronicity of the cultures. The main features of this sub-population, as evaluated by electron and fluorescence microscopy, were the following: 1. the presence in darkness of differentiating proplastids that were red fluorescent under UV, positive to TCNBT cytochemical reaction (specific for PSII) and negative to DAB (specific for PSI); 2. the acceleration of proplastid differentiation during the first 20–30 h of illumination; 3. the occurrence in both culture conditions of concentric lamellar bodies (LB_S). These structures were considered to be proplastids blocked in the first step of evolution, since they emitted a red fluorescence, were contained within compartments limited by a triple-layered envelope, were reactive to TCNBT in darkness and to both TCNBT and DAB in light conditions. Even if the action mechanism of 5-azaC on plastidogenesis in Euglena remains to be defined, the induced stimulatory effect on plastid differentiation pointed to a relationship between DNA methylation and plastid development. Furthermore, the presence of LB_S opens the possibility of studying early aspects of plastid development in Euglena.     

Changes of Thylakoid Membrane Stacks and Chl a/b Ratio of Chloroplast from Sacred Lotus (Nelumbo nucifera) Seeds During Their Germination Under Light

Changes of chloroplast thylakoid membrane stacks and Chl a/b ratio in the plumule of sacred lotus (Nelumbo nucifera Gaertn) seeds during their germination under light were as follows: Before germination there were giant grana and very low Chi a/b ratio (0.9) in the chloroplasts. Two days after germination, the thylakoid membranes of the giant grana gradually loosened and even destacked (disintegrated), the Chl a/b ratio was 1.06. Four clays after germination, the newly formed grana thylakoid membranes were 3–5 times shorter than those of the supergrana thylakoid membranes before germination and less grana stacks were seen; the Chl a/b ratio was 1.42. Six days after germination, the stacked thylakoi membranes became more orderly arranged. In addition the grana increased in number, the stroma thylakoid membranes were scarce, the Chl a/b ratio was 2.16. Eiglt days after germination, the thylakoid membranes in each granum decreased, but the total number of grana increased only slightly. In the meantime, some large starch grains and more stroma thylakoid membranes appeared; the Chl a/b ratio was 2.77. Ten days after germination normal thylakoid membrane structure was formed both in grana and stroma lamellae. They were arranged orderly as in the chloroplasts of other higher plants; the Chl a/b ratio was 2.80. The following conclusions could be drawn from the above mentioned results: 1) There was a negative correlation between the degree of stacking of the grana thylakoid membranes and the Chl a/b ratio. This statement further proved that the membranes stacking might mainly be induced by LHCII. 2) Development of the grana thylakoid membranes within chloroplasts from sacred lotus plumule followed that of the stroma thylakoid membranes, and the tendency of changes of their Chl 2/b ratio being from the lowest to the highest and then to normal were quite different from those of other higher plants. The chloroplasts iri the latter plants contain long parallel stacks of nonappressed primary thylakoids at second step, and the changes of their ratio of Chl a/b tend to be from the highest to the lowest and then to normal. There are indications that sacred lotus plumule might employ a distinctive developing pathway. This provides an important basis for Nelumbo to possess an unique position in phylogeny of Angiospermae.