Tissue homogenates, etioplasts, and developing chloroplasts were prepared from cucumber (Cumucis sativus L.) cotyledons in tris-sucrose. They were incubated aerobically in the dark or in the light at pH 7.7 in the presence or absence of a cofactor mixture containing coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate. These cofactors were previously shown to be essential for protochlorophyll and chlorophyll biosynthesis. Ultrastructural changes were monitored by electron microscopy. The following observations were made. (a) Crude homogenates contained agents which degraded etioplasts and developing chloroplasts. (b) Added cofactors were essential for the maintenance of the membrane structure; they were also implicated in the transformation of the prolamellar body in the absence and presence of light. (c) Light pretreatment of the cotyledons improved the maintenance of the developing chloroplast membranes during subsequent in vitro incubation. (d) In the presence of the cofactors, grana formation appeared to take place in the absence of nuclear-cytoplasmic control.

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.

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.

Vegetative cells of the juvenile (Chantransia) and adult forms of Batrachospermum were examined by thin-section and freeze-etch electron microscopy. The cells showed many features characteristic of the Rhodophyta. In addition, some aspects of red algal fine structure not closely examined previously are discussed. Lomasomes were most prevalent in association with the developing septum of dividing cells and at the distal end of apical cells. Vacuole formation involved expansion of portions of the endoplasmic reticulum and fusion of dictyosome vesicles with developing vacuoles. Two functionally different types of dictyosomes were observed: one contributed vesicles lacking a fibrillar content to the vacuole, whereas the other contributed vesicles with a fibrillar content to the cell wall. The development and structure of pit connections were similar to that described for other red algae. However, in addition to the usual plug structure, an amorphous plug cap and associated network of endoplasmic reticulum were observed. A possible role of this endoplasmic reticulum in intracellular transport is considered. Inclusions consisting of whorls of lamellae (concentric bodies) were seen in thin-section and freeze-etch. They appeared to originate in the cytoplasm and were released into the cell vacuole. Freeze-etched chloroplast thylakoids fractured to reveal two inner membrane faces, but in no case did the fracture reveal membrane surfaces bearing phycobilisomes.

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.

Colonies of Nostoc sphaericum were incubated in potassium tellurite and tetranitro-blue tetrazolium solutions with and without succinate substrate in the presence and absence of light. In all cases there is evidence that the reduction products are associated with the photosynthetic lamellae. Deposition of reduced tellurite with succinate substrate in the absence of light would rule out the reduction caused by photosynthesis (redox reaction). It seems, therefore, that the so-called photosynthetic lamellae in blue-green algal cells also function as mitochondrial equivalents. There is indication that reduced tellurite is continuously removed from the reduction site while TNBT-formazan is relatively immobile. There is no evidence for the reduction of tellurite on the plasma membrane or any of the cytoplasmic granules.

The application of sublethal doses of 3‐amino‐1,2,4‐triazole (AT) to germinating, light‐grown wheat grains causes chlorosis of the resulting leaves. An ultrastructural examination of the leaf tissue reveals that the plastids lack normal grana‐fret membrane systems and chloroplast ribosomes. A few disorganized membranes are always present in these chloroplasts. However, AT‐treated, dark‐grown seedlings contain proplastids with non‐crystalline prolamellar bodies and ribosomes. When these etiolated, treated plants are exposed to 600 ft‐c light for various periods of time, the proplastids fail to develop into normal, grana‐containing chloroplasts.

Ultrastructural studies ofthechloroplas,ts ofthenormal, yellow-green, andpale greenphenotypes ofZeamaysL.indicate thatthcinternal membranesystemiscontinuous withtheplastid envelop. The intramembraneous spaces, loculi, andfretchannels arealso continuous withinnercomponent oftheplastid envelop. Highenergycompounds orother photosynthates, formed inthegranaorfrets arethusseparated frombothstromaandcytoplasm byasingle membrane, either thefretmembrane ortheoutercomponent oftheplastid envelop. Sincethistypeofplastid ultrastructure isapparently foundonlyinplants exhibiting theHatch andSlackpathways ofphotosynthesis theremay bea relation betweenplastid ultrastructure andthepathways ofphotosynthetic carbonfixation.

From a gross morphological view, picloram (4-amino-3,5,6-trichloropicolinic acid) acted much like 2,4-D. As soon as 2 hr after treatment with higher concentrations of picloram (7.2 μg/plant), the stems began to swell and bend, and the treated leaves began to shrivel. Apical growth was stopped if concentrations were too high (72.0-7.2 μg picloram per plant). With lower concentrations (less than 0.72 μg picloram per plant), however, apical growth was only retarded and flowering delayed. The light-microscope study of the aerial portions of the plant revealed that the vascular cambium and its initials remained meristematic when affected by picloram. Initial divisions were periclinal, but later divisions were anticlinal and transverse, forming an adventitious root. As the adventitious root developed, the cortical cells were torn around the area of the root, and the epidermis was stretched until fissures developed.

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.

Chloroplasts, bothin situ and isolated, were dehydrated at low temperatures to avoid chlorophyll extraction. These chloroplasts were fixed with glutaraldehyde only or with glutaraldehyde followed by osmium. Chloroplasts dehydrated at low temperatures have narrower fret membranes, but the same dimensions of the partitions as chloroplasts dehydrated by standard techniques. However, the two membranes forming the partition are separated by an electron transparent region or A-component in low temperature dehydrated chloroplasts post-fixed with osmium. With glutaraldehyde only, the A-component appears as a thin discontinuous dark line. The results indicate that the A-component is a structural component of the partition and supports the hypothesis that chlorophyll and the hydrophibic lipids are localized there.

INTRODUCTION been carried out with anthocyanin as a marker of vacuolar material and with various enzymes as markers of mitochondrial and hyaloplasmic commethod of isolating cell organelles (2) and its markers of mitochondrial and hyaloplasmic commodification for use in isolating chloroplasts (3, ponents 5, 13), it has been used extensively in the study visual observations of the purity of these preparaof the intracellular distribution of water-soluble tions and the structure of these plastids have enzymes, minerals, and photosynthetic reaction been confined to light microscopy (16). Where products (4, 6-16). electron microscopy has been used, the isolated Tests of the purity of isolated chloroplasts have plastids generally have been prepared for study

Ultrastructural studies of the chloroplasts of the normal, yellow-green, and pale green phenotypes of Zea mays L. indicate that the internal membrane system is continuous with the plastid envelop. The intramembraneous spaces, loculi, and fret channels are also continuous with inner component of the plastid envelop. High energy compounds or other photosynthates, formed in the grana or frets are thus separated from both stroma and cytoplasm by a single membrane, either the fret membrane or the outer component of the plastid envelop. Since this type of plastid ultrastructure is apparently found only in plants exhibiting the Hatch and Slack pathways of photosynthesis there may be a relation between plastid ultrastructure and the pathways of photosynthetic carbon fixation.

The application of sublethal doses of 3-amino-1, 2,4-triazole (AT) to germinating wheat seedlings results in the formation of alibinistic leaves. These leaves grow and develop nearly as well as the control leaves for periods up to 1 week following germination (9). An tultrastrutctural examination of this tissue revealed that the chloroplasits were the only subcellular organelles altered morphologically by AT treatment. These altered chloroplasts lacked normal grana and fret membranes, but rather contained a few disorganized or concentric arranged membranes (2). In this study, we examined the effect of AT on the ribosomal composition of light-grown wheat leaves and found that the 70S chloroplastic ribosomes and 18S Fraction I protein of the chloroplast were absent. In addition, we confirmed earlier investigations (4, 5, 6, 8) which indicated that the chloroplasts contained only 70S ribosomes. Abotut 15 wheat grains (Triticum vulgare L. var. Seneca and Federation) were germinated in a petri dish containing 10 ml of 0.1 mm AT or distilled water. The plants were grown tunder 1000 ft-c of light (16 hr photoperiod, 21?) or in darkness. Following germination, distilled water was tused for the required watering and shoots were harvested on the seventh day and prepared for either ultrastructutral examination or sedimentation sttudies. For sedimentation studies, approximately 10 g of fresh leaf tissue were chilled and grouind in a mortar and pestle (2?) with an eqtual weight of stucrose-tris btuffer at pH 8.4 (7). The homogenate was strained throuigh 2 layers of cheesecloth, centrifuged for 30 mintutes at 23,000 X g (max, 1?) in a Servall SS-34 head and the suipernatant material was then centrifuged for 1.5 houirs at 226,000 X g in a Spinco 50 Ti head. The pellet was resuispended in a buiffer (pH 7.5) composed of 5 mM tris (Sigma) 7 mm magnesitum acetate and 5 mm mercaptoethanol, and clarified by centrifuging for 10 minuites at 8000 X g in an SS-34 head. The supernatant solution was removed and tused as the ribosomal suspension for the ultracentriftugal analysis on a Spinco MIodel E ultracentrifuge using a standard 12 mm, 40 sector cell in an AnD rotor. For electron microscopic stuidies, fresh tissue was fixed in 6 % glutaraldehyde (9 hr, 40) and embedded in Maraglas. Sections were cut with glass knives and double post-stained with aqueous uranyl acetate and lead citrate (3). Figure 1 shows a part of a chloroplast from an untreated, light-grown plant. The stroma (S) of this chloroplast contains many 170A particles (PR) which conform to the electron microscopical criteria for ribosomes since they were preserved by glutaraldehyde and osmium tetraoxide, strained 'with turanyl acetate and digested by ribonuclease (3). In contrast, a section of a plastid from an ATtreated plant (fig 2) showed that the stroma lacked ribosomal particles while the cytoplasm contained an abundance of ribosomes (CR). The ultracentrifugal pattern obtained with the ribosomal extracts from tissuie identical to those tused in the ultrastructural study is shown in figure 5. In this figuire, the direction of sedimentation was from left to right. Extracts from control, lighit-grown leaves (lower curve) showed 3 peaks with approximate sedimentation coefficients of 18S, 70S, and 80S and they represent Fractions I protein, chloroplastic and cytoplasmic ribosomes, respectively (4,5,8). In contrast, extracts of ATtreatedl, light-grown leaves (upper curve) yielded only an SOS peak, and the 18S and /OS peaks were completely absent in 5 separate experiments. In this sttudy, corrections were not made for viscosity effects duie to residual stucrose and protein concentration, therefore, the sedimentation valves are not exact. The comibined results of the tultrastructuiral and utltracentrifugal stuidies show that the treatment of light-grown, germinating seedlings with AT catused the complete loss of chloroplastic ribosomes and Fraction I protein but not cytoplasmic ribosomes. In contrast to the stuidies involving light-grown plants, AT treatment did not appear to alter the 1 Supported by grants from the USPHS (6F2HD-23, 340-OlAl) and the American Cancer Society.

The ultrastructure of the plastids from the genetically caused but maternally transmitted mutant iojap of maize was studied at four stages of development. The plastids of green and potentially green tissue were normal at all stages studied. The plastids of the white tissue were aberrant at all stages studied and lacked the normal grana-fretwork system as well as a normal prolamellar body. DNA-like fibrils were present in aberrant plastids, but ribosomes were absent. This indicates that chloroplast ribosomes are important in chloroplast membrane formation. Aberrant plastids fail to develop normally and are not a degeneration of normal plastids. Aberrant and normal plastids occur in single cells in green tissue, but only aberrant plastids have been found in white tissue.

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.