THE FINE STRUCTURE OF CHLOROPLASTS FROM MINERAL-DEFICIENT LEAVES OF PHASEOLUS VULGARIS

Thomson , W. W., and T. E. Weier . (U. California, Davis.) The fine structure of chloroplasts from mineral-deficient leaves of Phaseolus vulgaris. Amer. Jour. Bot. 49(10): 1047–1055. Illus. 1962.—An electron microscopic study of the changes in chloroplast structure as affected by the stress of nutrient deficiencies is described. Each deficiency produces characteristic changes in the ultrastructure of the chloroplast. In phosphorus and potassium deficiency the plastids develop fully before changes occur; then the grana break down into diffuse, electron-dense masses, forming a highly ordered lamellar system. The plastids of plants low in nitrogen and magnesium do not reach full development before changes occur. In nitrogen-deficient plastids, the stroma is greatly diminished and the grana compartments are swollen and reduced in number. In magnesium deficiency, the grana-fretwork system becomes disorganized and many star-bodies are formed. The absence of zinc blocks the full development of a grana-fretwork system, and large vacuoles are formed in conjunction with grana compartments.     

THE ULTRAMICRO STRUCTURE OF STARCH-FREE CHLOROPLASTS OF FULLY EXPANDED LEAVES OF NICOTIANA RUSTICA

Weier , T. Elliot . (U. California, Davis.) The ultramicro structure of starch-free chloroplasts of fully expanded leaves of Nicotiana rustica. Amer. Jour. Bot. 48(7): 615–630. Illus. 1961.—The grana of starch-free chloroplasts of fully expanded leaves of Nicotiana rustica are distinct, compartmented, subplastid entities. They vary in size, shape, orientation and in the distinctness with which their compartments are delineated. It has not been possible to equate accurately their micro and ultramicro appearances. At the ultramicro level, the grana are connected with each other at irregular intervals by a system of anastomosing channels. The partitions forming the compartments of the grana may be coarse or very fine but are constant in appearance in any given chloroplast. The loculi enclosed by the partitions may vary in size with a granum, depending upon their location or upon the physiological activity of the chloroplast. The stroma does not penetrate the grana; it may be relatively fluid and the grana-fretwork system may move within it. A double envelope, which may have pores connecting stroma and hyaloplasm, surrounds the chloroplasts. Materials may collect between the surfaces of the envelope. There is considerable variation in the ultramicro details of chloroplast structure of Nicotiana rustica. It is not yet possible to distinguish accurately between those variations which may be of physiological significance and those which may be induced by processing.     

THE GRANA OF STARCH-FREE CHLOROPLASTS OF NICOTIANA RUSTICA

The grana of chloroplasts of starch-free leaves of Nicotiana rustica are described in detail. Leaf sections were fixed in 2.5 per cent KMnO₄ and embedded in mixtures of butyl and ethyl methacrylate. Chain length of the polymer was modified by use of a transfer agent. The grana are composed of compartments consisting of electron-scattering partitions and electron-transparent loculi. Compartments are not open to the stroma so that the grana are distinct subplastid organelles. Adjacent grana are connected by an anastomosing fretwork system composed of flexuous channels bordered by electron-scattering membranes. Ten different kinds of granum margins are described. These marginal variations depend upon grana-fretwork connections and internal marginal connections between adjacent loculi. A study of serial sections suggests, at least in some plastids, the occurrence of a possible orderly spiral arrangement of compartment-fretwork connections. Adjacent grana may have common compartments. Grana may branch. Variations in shape may depend upon the angle the section bears to the axis of the cylinder. This should also influence the relative thickness and sharpness of the partitions. Since all shapes and variations in partition thickness and sharpness cannot be accounted for on the basis of the orientation of the grana, such variations probably occur naturally. Grana vary in size, ranging from those few which have a single partition to those having 50 or more compartments which extend completely through the width of a plastid. Relationships between grana of different sizes and between compartments and frets indicate the possibility of growth of grana from union or extension of compartments and formation of compartments from the union of frets.     

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.     

The inhibition of kohlrabi chloroplast degeneration by kinetin

Summary Detached kohlrabi leaves of late autumn material yellowed completely after 6 days in weak light. This process was accompanied by a decrease in chlorophyll, protein, and ribonucleic acid levels. In the chloroplasts, degeneration symptoms such as reduction in chloroplast volume, the decay of grana, development of the long thylakoid system, disappearance of chloroplast ribosomes, increase in the volume of plastoglobuli, and finally a complete breakdown of plastids in the digesting vacuoles, were observed. The ultrastructural changes in degenerating kohlrabi chloroplasts resembled those described earlier for brussels sprouts (Dennis et al. 1967), which suggests that the plastid degeneration model may be specific for speciesBrassica oleracea L.Kinetin inhibited the fall in the level of chlorophyll, proteins, and RNA in relation to the control material, and even stimulated chlorophyll and protein synthesis to a level higher than that of the initial material. Treatment with kinetin also markedly delayed the loss of chloroplast ribosomes. The most evident effect of the kinetin influence on the plastid ultrastructure was the stimulation of the formation and maintenance of grana. A possible mechanism for these processes in the light of the recent studies on the chloroplast membranes is discussed.     

ULTRASTRUCTURAL CHANGES OF CHLOROPLASTS IN ATTACHED AND DETACHED,AGING PRIMARY WHEAT LEAVES

Degradation of chloroplasts is shown in mesophyll cells of primary leaves of wheat. The sequence of ultrastructural changes in chloroplasts of naturally senescing leaves is compared with that of detached, aging leaves. In chloroplasts of naturally senescing leaves, the first indications of aging are the appearance of osmiophilic globuli and reorientation of the thylakoidal system. The membranes of the grana and intergrana lamellae then become distended and later dissociate into distinct vesicles. Concurrent with these membrane changes, osmiophilic globuli increase in size and number, and the stroma breaks down. Finally, the chloroplast envelope ruptures and plastid contents disperse throughout the cell's interior. In chloroplasts of mesophyll cells in detached, aging leaves, initial changes also include appearance of osmiophilic globuli, but later stages of chloroplast degradation are different. The chloroplast envelope ruptures before the lamellae break down. Swelling of grana and intergrana lamellae is not pronounced and, additionally, the thylakoidal system degenerates without forming vesicles or numerous osmiophilic globuli. These differences in the sequence of chloroplast degradation indicate that naturally senescing leaves rather than detached, aging leaves should be used in studies of chloroplast senescence.     

THE CHLOROPLAST STRUCTURE OF IOJAP MAIZE

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.     

DIFFERENTIATION OF THE CHLOROPLAST OF ANTHOCEROS

It has been demonstrated that the following changes accompany differentiation in the plastid of the liverwort Anthoceros eckloni. The inner membrane of the plastid folds to give rise to small vesicles which grow and may fuse to form thylakoids, i.e. flat bags. The thylakoids may "pair" to produce the doublets (thick membranes) of the grana. The doublets may be produced also by the invagination of a thylakoid. In both cases, the doublets are produced only where outside-to-outside contact of thylakoid membranes occurs, which supports the thesis that the thylakoid membranes are polarized. The thylakoids also fold outwards, anastomose, and may fuse. This results in a complicated membrane system, for which an interpretation becomes very difficult. The starch is produced in the matrix, and the pyrenoid bodies are interpreted as specialized regions of the matrix. Younger plastids have grana, but the mature plastid has so many doublets that distinct grana cannot be recognized. This interpretation of the changes which occur during the differentiation of the plastid differs radically from those of Menke (1961) and Manton (1962) who studied this same genus, but is compatible with findings in algae and angiosperms.     

ULTRASTRUCTURAL CHANGES IN SUNFLOWER CHLOROPLASTS FOLLOWING INOCULATION WITH PSEUDOMONAS SYRINGAE PV. TAGETIS

Severe chlorosis and ultrastructural modifications of chloroplasts occur in sunflower in response to infection by Pseudomonas syringae pv. tagetis. Chlorosis became apparent within 2 days after the cotyledons of 10-day-old sunflower seedlings were inoculated with the bacteria. The first symptoms generally appeared in the center of leaves at the second node above the cotyledons. Leaves above the second node lost essentially all of their pigmentation but remained turgid and continued to expand. Grana thylakoids became dilated and separated from the granal stacks. These thylakoid membranes did not chemically breakdown as in the case in chromoplast formation or normal chloroplast senescence. Both grana and stroma thylakoid membranes coalesced to form a large membrane sheet within the plastid. The ultrastructural changes are unlike those reported to be caused by other chlorosis-inducing bacteria or chlorosis associated with normal senescence.     

THE CUP PLASTID OF NICOTIANA RUSTICA

Weier , T. E., and C. R. Stocking . (U. California, Davis.) The cup plastid of Nicotiana rustica. Amer. Jour. Bot. 49(1): 24–32. Illus. 1962.—In situ and isolated chloroplasts of Nicotiana rustica have been studied by light and electron microscopy. Under certain conditions, notably of low light intensity, the starch-free plastid forms a cup. In isolated plastids the form may be modified by the tonicity of the isolation medium. In situ, the cup always faces the cell wall. Electron micrographs show the cup to be formed of compartmented grana connected at irregular intervals by flexuous channels known as frets. The interior of the cup is filled with a finely granular stroma which also forms the material surrounding the grana and the frets in the body of the cup. The grana radiate outward from the central stroma. They may be considered as cylinders. They are fairly rigid, as curvatures to form the cup-shape occur only in the interconnecting fretwork. The compartments may have a limited movement with reference to their axis. These evidences of movement of the part of the ultramicro plastid structures thought to contain chlorophyll suggest that the movement may be related to changes in light intensity or other factors influencing the rate of photosynthesis.     

SUBMICROSCOPIC STRUCTURE AND DEVELOPMENT OF THE CHLOROPLASTS OF PSILOTUM TRIQUETRUM

Sun , C. N. (Washington U., St. Louis, Mo.) Submicroscopic structure and development of the chloroplasts of Psilotum triquetrum. Amer. Jour. Bot. 48(4): 311–315. Illus. 1961.—Aerial stems and stem tips of Psilotum triquetrum were used for the study of the fine structure and development of chloroplasts. The chloroplasts of Psilotum are ellipsoidal, with a principal axis of approximately 13 μ and a short axis of approximately 3.6 μ. They are bounded by a well-defined outer membrane which consists of 2 layers. Within the laminar system of the stroma, the lamellated grana appear as sharply defined regions. The grana are about 1–1.6 μ in diameter. They are distributed more or less uniformly throughout the entire chloroplast with the exception of a very narrow peripheral zone. Relatively large, osmiophilic globules occur in groups in the stroma. The development of the Psilotum chloroplast may be summarized as follows: (1) in the undifferentiated proplastid, vesicles occur; (2) lamellated layers are formed by the fusion of vesicles; (3) the lamellae multiply by a process of thickening and splitting; (4) the grana are differentiated within a certain area by heterogeneous deposition of material and by further cleavage of the lamellae. Osmiophilic globules are present throughout the developmental stages, and increase in number and size with increase in age of the chloroplast.     

THE STRUCTURE OF GRANA IN FLOWERING PLANTS

A model of granum structure, consisting of multiple helical frets wound around each cylindrical granum, is proposed as a representation of granum structure for flowering plants in general. This model was originally formulated for grana in the mesophyll plastids of Zea mays and was subsequently extended to grana of Phaseolus vulgaris by additional studies. The model is now shown to be applicable also to grana in the plastids of Cannabis sativa, Elodea canadensis, Nicotiana rustica, Pisum sativum, and Spinacia oleracea. This sample of seven angiosperms includes genera commonly studied by other workers in their ultrastructural investigations of plastid structure.     

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.     

Structural and Functional Changes of Mesophyll Cells during Leaf Growth in Two Species of Spring Ephemers

The chloroplast ultrastructure, plastid pigments, and potential photosynthesis of leaf mesophyll cells were examined during the vegetative season of two spring ephemers Scilla sibirica Haw. and Chionodoxa luciliae Boiss. The development of chloroplasts was shown to precede the appearance of photosynthesis. The earliest stage of leaf growth was marked by the synthesis of carotenoids that play a structural and organizational role in the formation of chloroplast grana and protect the photosynthetic apparatus from photodynamic destruction under high insolation and low temperature conditions. Chlorophyll synthesis was closely correlated with the dynamics of potential photosynthesis. All these structural and functional features of mesophyll cells reflect the evolutionary strategies of adaptation in spring ephemers, which enable these plants to complete their short life cycle in the environment combining low temperature and high insolation.     

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 and Ultrastructure in Yellowish Mutants of Sugar Cane

The comparisons of the ultrastructures of plastids in yellowish mutant and yellowish-green striped mutant and in normal green plant from tissue culture of sugar cane were made. There was no difference found in the structure and development of chloroplasts between the normal green leaves and the green tissue of striped leaves, but the plastid in the yellowish tissue of two kinds of mutants were anomalous. They had not a fully developed system of grana and stroma lameilae as in the normal green leaves. This aberrant plastid contained only some vesicles, a few lamellae and more or less clearly defined ribosome particles and DNA-like mierofibrils, while some stacking and swelling of thylakoids were often observed. In some sections of this aberrant plastid a bunchy lamellae and cross connective fibrils between parallel lamellae were often found too. However, the mixed cell which contains both of normal chloroplast and defective plastid together was never found in the leaves of the mutant plants. It was suggested that yellowish and yellowish-green striped leaves from tissue culture of sugar cane might be caused by nuclear gene mutation.     

Chloroplast-ultrastructure and chlorophyll content in leaves from Quercus branches with and without epiphytic lichen thalli

Abstract The effect that the massive presence of lichen thalli growing on the branches of Quercus pyrenaica and Q. rotundifolia leaves has on their chloroplasts been studied. In both species there were significant decreases in the amount of chlorophylls in the leaves of twigs with a dense cover of lichens in comparison with the leaves from thallus-free twigs. The areas and perimeter of chloroplasts in leaves from twigs with epiphytes did not differ significantly from those in leaves without epiphytes. However, in leaves with epiphytes the percentage of chloroplast area occupied by starch was higher. In Q. pyrenaica the number of grana per chloroplast section and per μm², the percentage of chloroplast stroma occupied by grana, the average number of thylakoids forming grana and the grana width was significantly smaller in leaves near lichen populations. These results are discussed and related to the great chelating capacity of the lichen's substances.     

THE STRUCTURAL RELATIONSHIPS OF THE INTERNAL MEMBRANE SYSTEMS OF IN SITU AND ISOLATED CHLOROPLASTS OF HORDEUM VULGARE

Healthy chloroplasts of Hordeum vulgare are compared with chloroplasts subjected to abnormal stresses such as in situ disruption, isolation, isolation plus washing in 0.5 m sucrose, and isolation plus washing in 0.5 m sucrose and distilled H₂O. Normal chloroplasts resemble those of Nicotiana rustica and Phaseolus vulgaris in being composed of compartmented grana connected by an anastomosing fretwork system. They differ in having a somewhat greater incidence of parallel frets and double partitions. Under conditions of stress both grana and fretwork undergo varying degrees of swelling, and the double partition maintains its structural integrity. Grana are more resistant to abnormal stresses than the fretwork. Fret connections with more than 3 grana do not generally occur, but in some micrographs a single pathway may be traced through several grana. Washing isolated chloroplasts in distilled water results in an enlargement involving compartments of 2 or more grana together with the associated fretwork membranes. These results indicate that the grana in mature chloroplasts of Hordeum vulgare, like those of Nicotiana rustica and Phaseolus vulgaris, are compartmented structural units and not a series of localized aligned thickenings in regular extensive discs. These enlargements are complex structures comprising the membranes and spaces of both grana and frets. The swelling indicates an increase of locular and fret channel substance and possibly an enlargement of membrane surfaces. Dried down on grids, the compartments and frets appear as flat discs with radial appendages.     

PLASTID ORGANIZATION IN PHENOTYPICALLY GREEN LEAF TISSUE OF A GENETIC ALBINO STRAIN OF NICOTIANA (SOLANACEAE)

Plastid organization within phenotypically green leaf tissue that forms in albino plants of a genetic albino strain of Nicotiana has been examined with the transmission electron microscope. Studies revealed the presence of plastids with and without thylakoids. When present, thylakoids were loosely and irregularly scattered in the stroma or organized either into several large spindle-shaped grana or into a single compound granum with deeply indented margins. Plastids without thylakoids were vesiculated and resembled the typical genetic albino type. Plastid types were not segregrated into individual cells and no plastid type appeared to be typical for the mutant tissue. Orientation of grana and thylakoid membrane associations were noted as well as the presence of osmiophilic globules, starch grains and DNA-like fibrillar areas.     

Methylation of chloroplast DNA does not affect viability and maternal inheritance in tobacco and may provide a strategy towards transgene containment

We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background.