ULTRASTRUCTURE OF THE SHOOT APEX OF TOMATO (LYCOPERSICON ESCULENTUM)

The vegetative shoot apical meristem of tomato (Lycopersicon esculentum Mill.) was examined at the ultrastructural level. The meristem consisted of a surface layer that was different from the rest of the meristem and was unique among the dicotyledonous species. The cells of the surface layer contained large distal vacuoles with relatively large electron-dense inclusions, proplastids with membrane-bound inclusions (MB), and differentiating chloroplasts. In addition, periclinal and oblique divisions were observed in the surface layer cells along with anticlinal divisions. The cells of the subsurface layers contained small vacuoles with fewer inclusions as well as proplastids of various shapes but without MB. Differentiating chloroplasts were not observed in these cells, but autophagic vacuoles at various stages of development were present. The normal complement of cell inclusions, e.g., the mitochondria, golgi bodies, endoplasmic reticulum (ER), ribosomes, and microtubules were observed in subsurface layers, and in many cells the ER was observed to be continuous with the outer membrane of the nuclear envelope and with the plasmalemma. Further below in the meristem, cells contained both the proplastids and differentiating chloroplasts with MB. In the latter, the outer membrane of the MB was found to be continuous with the developing lamellae, suggesting that MB probably serve as the storage centers for lamellae membranes. Near the base of the meristem, in the pith-rib meristem, enlarged cells containing large vacuoles and differentiated chloroplasts were present.     

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.     

甜菊组织培养物中叶绿体的超微结构与脱分代

含有叶绿体的甜菊（Ｓｔｅｖｉａｒｅｂａｕｄｉａｎａ）愈伤组织细胞转移至新鲜培养基后,导致光合片层的逐渐减少或消失,最后叶绿体脱分化形成原质体样的结构。超微结构观察表明,光合片层的减少或消失与降解及叶绿体分裂特别是不均等缢缩分裂而致基质组分和类囊体膜稀释有关。这一过程并不完全同步,一些质体含有少量正常的片展而另一些质体含有退化的片层甚至片展结构完全消失。细胞的一个明显特点是细胞器大多聚集在细胞核附近,细胞质增加并向细胞中央伸出细胞质丝。同时可观察到原质体。培养７ｄ后,许多细胞呈分生状态,细胞质富含细胞器,充满了细胞的大部分空间。此时细胞中的质体大多呈原质体状态。在细胞生长的稳定期,质体内膜组织成基质基粒片层,同时质体核糖体增加。文中讨论了高度液泡化细胞脱分化与细胞中叶绿体脱分化的关系。     

ULTRASTRUCTURAL CYTOLOGY OF CALLUS CULTURES OF STREPTANTHUS TORTUOSUS AS AFFECTED BY TEMPERATURE

Tissue culture cells of Streptanthus tortuosus var. orbiculatus (Cruciferae) which have acquired a spherical viruslike particle located in their nucleoli, designated cell line STV, developed supergranal chloroplasts and lost the ability to differentiate vascular tissues. The effect of temperature on the ultrastructural cytology of one line of the STV tissue, STV-I, was compared with the effect of temperature on the ultrastructural cytology of tissue culture cells lacking the viruslike particles (control cell lines). At 4 C, the cellular and ultrastructural appearance of control tissue culture cells differed from that of tissue grown at 22 C by producing increased amounts of endoplasmic reticulum and dictyosomes and by reduction of chloroplast thylakoids. STV-I cells were generally moribund as a result of 4 C treatment. Chloroplast thylakoids were also reduced in control tissue following growth at 10 C and the apparent quantities of endoplasmic reticulum and dictyosomes were similar to those observed in control cells grown at the control temperature (22 C), but less than those observed in tissue subjected to 4 C. STV-I tissue grown at 10 C demonstrated increased endoplasmic reticulum and dictyosomes and reduction of polysomal configurations. The mitochondrial morphology was variable and the cells contained supergranal chloroplasts and proplastids. At the control temperature (22 C), the fine structural appearance of control tissue culture cells was typical of parenchyma cells, but STV-I cells contained mitochondria of variable morphology and two types of chloroplasts— normal and supergranal. Control tissue grown at 30 C also contained proplastids, but these proplastids contained starch in contrast to the proplastids in control tissue grown at low temperatures. The ultrastructural cytology of STV-I cells grown at elevated temperature (30 C) was characterized by enlarged mitochondria containing massive lipid bodies and the presence of protoplastids with starch and supergranal chloroplasts.     

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.     

EFFECTS OF DARKNESS AND OF STREPTOMYCIN ON THE FINE STRUCTURE OF EUGLENA GRACILIS

Siegesmund , Kenneth A., Walter G. Rosen , and Stanley R. Gawlik . (Marquette (J., Milwaukee, Wis.) Effects of darkness and of streptomycin on the fine structure of Euglena gracilis. Amer. Jour. Bot. 49 (2) : 137–145. Illus. 1962.—Dark-grown Euglena gracilis cells, transferred from streptomycin (SM)-containing medium to SM-free medium 5 days before transfer to light, turn green normally, indicating that proplastids are unaffected by SM. SM-bleached cells, grown in light, contain numerous bodies composed of concentric lamellae (CL bodies). These differ from chloroplasts in that their lamellae lack electron-dense dots, are not coalesced, and they lack a 3-layered limiting membrane and pyrenoids. CL bodies are absent from dark-grown normal and dark-grown SM-bleached cells, as well as from light-grown normal cells. It is suggested that CL bodies result from a derangement of chloroplast synthesis caused by SM blockage of chlorophyll synthesis.     

细叶黄芪叶肉原生质体发育早期几种细胞器的变化

在细叶黄芪叶肉原生质体发育早期,细胞器的变化较大。离体培养4h后,线粒体的嵴和基质物质开始增加。培养3—5天后,线粒体的数量增加5倍以上,此时可见大部分线粒体围绕细胞核分布。在培养24h后,高尔基体开始发育,它们主要分布在细胞质周边区域。多糖细胞化学染色表明,高尔基体内沉积着大量嗜银物质。培养1天后,粗面内质网开始发育。培养3天时,部分叶绿体边缘出现一些空隙结构。随着叶绿体内膜结构的消失,淀粉粒增大,叶绿体逐渐转变为造粉质体。     

甜菊叶愈伤组织诱导过程中叶绿体的超微结构变化

观察了甜菊(Stevia rebaudiana Bertoni)叶外植体愈伤组织诱导过程中叶绿体的超微结构变化。结果表明,当叶外植体转移到培养基上培养后,叶绿体的片层结构逐渐退化。在叶绿体发生退化的过程中伴有叶绿体出芽和原质体的形成。推测新产生的原质体来自叶绿体产生的芽状体。而叶绿体本身最后完全解体消失。叶绿体超微结构的这种变化与高度液泡化的叶肉细胞脱分化至分生状态是平行的。随着培养的进行,分生状态的细胞发生液泡化变为薄壁细胞时,在愈伤组织表层的细胞中,质体重新形成片层结构,而内部细胞的质体则充满淀粉粒。     

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.     

Leucoplasts: a distinct kind of organelles lacking typical 70S ribosomes and free thylakoids

Non-pigmented plastids were observed in fully differentiated cells from leaves and stem tissues of various species. Although showing important differences in size and shape, these plastids exhibit permanent structural features which allow to get them together as a distinct kind of organelles: the leucoplasts. Leucoplasts are distinct from the proplastids and every intermediate stage of plastid differentiation, from white chromoplasts and tuber amyloplasts. Mature leucoplasts do not contain an autonomous central system of thylakoids structurally independent from the envelope and, therefore, are never green. However, the envelope inner membrane invaginates within the plastid a cisternal or tubular stroma reticulum connected with the intermembrane space of the envelope. In addition, the leucoplast stroma is often less dense than chloroplasts stroma and contain several nucleoids with DNA fibrils. However, 70S ribosomes either scattered in the stroma or attached to the stroma reticulum or the envelope are not visible in ultrathin sections of leucoplasts stained with uranyl and lead. The existence of more discrete particles as dense as ribosomes is suggested. The relationship between the absence of ribosomes and thylakoids is discussed. Except for their specific role in C10 monoterpene synthesis in glandular cells, the functions of leucoplasts in plant cells remains largely up to now a matter of conjecture.     

THE EFFECT OF LIGHT INTENSITY AND SUCROSE FEEDING ON THE FINE STRUCTURE IN CHLOROPLASTS AND ON THE CHLOROPHYLL CONTENT OF ETIOLATED LEAVES

Changes in the fine structure of proplastids of etiolated leaves exposed to various conditions of light and darkness for 24 and 48 hours were investigated, and the chlorophyll content of the leaves so treated was determined in vivo. The light treatments were given while the leaves were floated on tap water or on a 0.2 M sucrose solution. Leaves floated on water under low light intensity (2 foot-candles) were low in chlorophyll and contained plastids with concentric rows of vesicles. Transferring the leaves back to darkness resulted in the disappearance of the concentric rigs and re-formation of vesicular centers together with straight rows of vesicles and tubules, evenly spaced throughout the stroma. Chloroplasts of leaves floated on a sucrose solution under low light showed large vesicular centers together with stacks of rows of elongated tubules. The same chloroplast structure was found in leaves floated on a sucrose solution in the dark, after having been exposed to weak light for 24 hours. Chlorophyll content in these leaves was the same as in leaves floated on water under high light intensity, where the chloroplasts had normal grana and lamellae. The effect of the investigated factors on plastid development is discussed.     

Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves

Intact chloroplasts isolated from Euglena gracilis exhibit high rates of light-driven protein synthesis, whereas protein synthesis by isolated proplastids is absolutely dependent upon the addition of an exogenous energy source in the form of equimolar ATP and Mg²⁺. ATP and Mg²⁺ also stimulate translation by chloroplasts. The greatly increased rates of protein synthesis obtained by supplementing proplastids with ATP and Mg²⁺ have allowed the first clear characterization of proplastid translation products. Two-dimensional polyacrylamide gel electrophoretic analysis of proteins synthesized in organello shows that, while many translation products are common to both plastid types, most are unique to either the proplastid or the chloroplast. Pulse-chase experiments using both proplastids and chloroplasts indicate similar rates of turnover of newly synthesized proteins in both types of plastids. Thus, the differences seen between proplastid and chloroplast translation products are apparently not due to turnover. Immunoprecipitation of large subunit of ribulose-1,5-bisphosphate carboxylase (LS) from pulse-chase experiments indicates that LS is made in both proplastids and in chloroplasts and that the rate of LS turnover is similar in both types of plastids.     

Protein crystalloids in ribosome-deficient plastids of Aeonium domesticum cv. variegatum (Crassulaceae)

Protein crystalloids are typical constituents of Aeonium domesticum plastids. They are composed of hexagonally arranged tube-like elements situated in the stroma without a bordering membrane. The single tubule has an external diameter of about 20 nm and an internal one of about 10 nm. The green-white-green mesochimera Ae. domesticum cv. variegatum contains normal chloroplasts in the green tissue and colourless plastids in the pale tissue. The defective plastids have a double-layered envelope, scarce internal membrane structures and contain, in the mature stage, a large vacuole. Plastid ribosomes can be detected only rarely in proplastids. They lose their ribosome complement entirely in the course of development. Polyacrylamide gel electrophoresis of total nucleic acids extracted from white tissue revealed the absence of the 23S and 16S rRNA normally present in plastids. Despite the loss of ribosomes, the plastids contain large protein crystalloids, which are structurally identical with those of normal green chloroplasts. Consequences concerning problems of encoding and transport of crystalloid protein(s) are briefly discussed.Abbreviations CAM crassulacean acid metabolism - FIP fraction I protein - L I epidermis - L II subepidermal layer - L III leaf core - SPC succulent protein crystalloid This is the first part of a series on the crystalloid-forming succulent protein     

Studies on ferrochelatase. The enzymic formation of haem in proplastids, chloroplasts and plant mitochondria

1. Ferrochelatase was demonstrated in the chloroplasts and proplastids isolated from the primary leaves of beans (a dicotyledon) and oats (a monocotyledon). It was also detected in chloroplasts from etiolated bean seedlings made green by illumination before being harvested. The specific activities of the three types of bean organelles are similar, as are the specific activities of the oat proplastids and chloroplasts. 2. Chloroplasts from young spinach leaves also contain ferrochelatase; these chloroplasts were tested for their ability to form magnesium tetrapyrroles and found unable to catalyse the insertion of Mg(2+) into mesoporphyrin IX. 3. Ferrochelatase was also detected in potato tuber mitochondria. 4. Ferrochelatase activity in these plant preparations is much less stable on storage than similar preparations from bacteria and animal tissues. 5. Temperature affects the activities of spinach chloroplast ferrochelatase and rat liver ferrochelatase differently. Activity of the chloroplast enzyme increases as the temperature rises from 20.6 degrees to 26 degrees , but becomes increasingly inactivated as the temperature rises further to 38 degrees . The initial velocity of the mammalian enzyme, however, increases as the temperature rises from 25.8 degrees to 65 degrees , but the enzyme is inactivated after several minutes at 65 degrees .     

DAG, a gene required for chloroplast differentiation and palisade development in Antirrhinum majus.

We have identified a mutation at the DAG locus of Antirrhinum majus which blocks the development of chloroplasts to give white leaves with green revertant sectors. The green areas contain normal chloroplasts whereas the white areas have small plastids that resemble proplastids. The cotyledons of dark-grown dag mutant seedlings have plastids which also resemble proplastids. The palisade cells in the white areas of dag mutant leaves also lack their characteristic columnar shape. The DAG locus was cloned by transposon tagging: DAG encodes a novel protein with a predicted Mr of 26k, which is targeted to the plastids. Cleavage of its predicted transit peptide gives a mature protein of Mr 20k. Screening of databases and analysis of Southern blots gave evidence that DAG belongs to a protein family with homology to several proteins of unknown function from plants. Expression of DAG is required for expression of nuclear genes affecting the chloroplasts, such as CAB and RBCS, and also for expression of the plastidial gene RPOB encoding the plastidial RNA polymerase beta subunit, indicating that it functions very early in chloroplast development.     

Variation of plastid types in spinach

Summary During growth in the light the plastids of cultured leaf discs of spinach divide, increase in size, and differentiate in a similar manner to those in intact leaves. By contrast when l'eaf discs are grown in the dark prolamellar bodies begin to develop in partially differentiated chloroplasts within 2 hours. After 7 days growth in the dark the plastids contain many vesicles which appear to arise from swelling of thylakoids. These vesicles often contain large crystals. When dark grown discs are illuminated they regreen and fully differentiated chloroplasts are reformed.Proplastids are found in the stem apex of whole plants; these develop into the partially differentiated chloroplasts of young leaves, which divide and increase in size to form the fully differentiated chloroplasts of mature leaves. The cotyledons of dormant seeds contain proplastids; these develop into amyloplasts during germination and after exposure to white light differentiate into chloroplasts.     

PIGMENTS AND PLASTIDS IN CULTURES OF TOTIPOTENT CARROT CELLS

Cells from a strain of carrot which was prone to form deep-seated chlorophyll in its storage organ have been cultured in a manner that promoted them to organize into plantlets. Whereas the free cells contained only chloroplasts, the plantlets derived from these cells formed all types of plastids (“proplastids,” leucoplasts, chromoplasts, and chloroplasts) in accordance with the location of the cells in question in the developing plant body. The developmental history of the plastids has been traced with the electron microscope. The events of chloroplast development, previously described by Israel and Steward (1967) for cultured carrot explants, have been verified. The bearing of this new evidence upon the control of plastid development and biochemistry is discussed and related to other recent studies. The conclusion is that all totipotent carrot cells have plastids as essential organelles but that their final form and content are sharply defined by the factors inherent in the location of the cells in the plant body as it emerges.     

Chloroplast Development in Rye Coleoptiles

In the parenchyma cells of 1-d-old dark-grown rye coleoptiles (Secale cereale) proplastids occurred which sometimes contained starch grains. During coleoptile growth in darkness starch-filled amyloplasts are formed from the preexisting proplastids. No prolamellar bodies were observed in the stroma of the plastids of the etiolated coleoptile. After irradiation of 3-d-old etiolated coleoptiles with continuous white light three different types of plastids occurred. In the epidermal cells proplastids were observed. The parenchyma cells below the stomata of the outer epidermis (above the two vascular bundles) contained mature, spindle-shaped chloroplasts with a well-developed thylakoid system. In the parenchyma cells that surround the vascular bundles amyloplasts with some thylakoid membranes (chloroamyloplasts) occurred. The mesophyll cells of the primary leaves of dark-grown seedlings contained etioplasts with large prolamellar bodies. In the primary leaves of irradiated plants chloroplasts similar to those of the parenchyma cells of the coleoptile were observed. Our results show that the rye coleoptile, which grows underground as a heterotrophic organ, is capable of developing mature chloroplasts upon reaching the light above the soil surface. The significance of this expression of photosynthetic capacity for the carbon economy of the developing seedling is discussed.