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.     

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.     

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.     

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 Ultrastructure of Phaseolus vulgaris Chloroplasts

A comparison of bean chloroplasts after being fixed in potassiumpermanganate, osmium, and formaldehyde coupled with negativestaining shows that the general organization of the chloroplastis similar in all cases. However, the mature chloroplasts ofbean vary considerably in the extent and orientation of theinternal membranes—the grana and the interconnecting membranesbetween the grana. The interconnecting membranes are thin, branching,flexuous structures. This is illustrated by serial sectionsand by cross- and face-view sections of osmium- fixed chloroplastsand the best model, which allows for a considerable flexibilityin the orientation of the grana and also describes the thininterconnecting membranes between the grana, is the grana-fretworksystem proposed by Weier (1961). Furthermore, the comparative studies show that the internalregions of grana are separated from the stroma and that thegrana-fretwork systems appear to be a continuous membrane system.This membrane is single along the frets, end compartments, andgranal margins. It is double in the partitions of the granabut separated by a component, possibly a cementing material,which does not stain. It is suggested that this membrane isstructurally similar in the chloroplasts of higher plants, butthat its overall organization may vary from one plant to another.     

Stimulation by Light of Rapid pH Regulation in the Chloroplast Stroma in Vivo as Indicated by CO2 Solubilization in Leaves

Leaves of Brassica oleracea, Helianthus annuus, and Nicotiana rustica were exposed for 20 s to high concentrations of CO2. CO2 uptake by the leaf, which was very fast, was measured as a transient increase in the concentration of oxygen. Rapid solubilization of CO2 in excess of that which is physically dissolved in aqueous phases is proposed to be caused by bicarbonate formation in the stroma of chloroplasts, which contain carbonic anhydrase. On this basis, pH values and bicarbonate accumulation in the chloroplast stroma were calculated. Buffer capacities were far higher than expected on the basis of known concentrations in the chloroplast stroma. Moreover, apparent buffer capacities increased with the time of exposure to high CO2, and they were higher when the measurements were performed in the light than in the dark. During prolonged exposure of leaves to 16% CO2, calculated bicarbonate concentrations in the chloroplast stroma exceeded 90 mM in the dark and 120 mM in the light. The observations are interpreted as indicating that under acid stress protons are rapidly exported from the chloroplasts in exchange for cations, which are imported. The data are discussed in terms of effective metabolic pH control by ion transport, first across the chloroplast envelope and, then, across the tonoplast of leaf mesophyll cells. The direct involvement of the vacuole in the regulation of the chloroplast pH in leaf cells is suggested.     

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 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 Ultrastructure of Chloroplasts in Mineral-deficient Maize Leaves

The ultrastructure of mesophyll chloroplasts in full-nutrient and mineral-deficient maize (Zea mays) leaves was examined by electron microscopy after glutaraldehyde-osmium tetroxide fixation. Nitrogen, calcium, magnesium, phosphorus, potassium, and sulfur deficiencies were induced by growing the plants in nutrient culture. Distinctive chloroplast types were observed with each deficiency. Chloroplasts from nitrogen-deficient plants were reduced in size and had prominent osmiophilic globules and large grana stacks. Magnesium deficiency was characterized by the accumulation of osmiophilic globules and the progressive disruption of the chloroplast membranes. In calcium deficiency, the chloroplast envelope was often ruptured. Chloroplasts from potassium- or phosphorus-deficient plants possessed an extensive system of stroma lamellae. Sulfur deficiency resulted in a pronounced decrease of stroma lamellae, an increase in grana stacking, and the frequent occurrence of long projections extending from the body of the chloroplast. These morphological changes were correlated with functional alterations in the chloroplasts as measured by photosystem I and II activities. In chloroplasts of the nitrogen- and sulfur-deficient plants an increase in grana stacking was associated with an increase in photosystem II activity.     

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.     

Chloroplast Structure and Starch Grain Accumulation in Leaves That Received Different Red and Far-Red Levels during Development

An important step in understanding influence of growth environment on carbon metabolism in plants is to gain a better understanding of effects of light quality on the photosynthetic system. Electron microscopy was used to study chloroplast ultrastructure in developing and fully expanded leaves of tobacco (Nicotiana tabacum L. cv Burley 21). Brief exposures to red or far-red light at the end of each day during growth under controlled environments influenced granum size, granum number and starch grain accumulation in chloroplasts, and the concentration of sugars in leaf lamina. Far-red-treated leaves had chloroplasts with more but smaller grana than did red-treated leaves. Red light at the end of the photosynthetic period resulted in more and larger starch grains in the chloroplasts and a lower concentration of sugars in leaves. Chloroplast ultrastructure and starch grain accumulation patterns that were initiated in the expanding leaves were also evident in the fully expanded leaves that received the treatment during development. It appears that the phytochrome system in the developing leaves sensed the light environment and initiated events which influenced chloroplast development and partitioning of photosynthate to adapt the plant for better survival under those environmental conditions.     

Regreening of senescent Nicotiana leaves. II. Redifferentiation of plastids

Single senescent leaves attached to decapitated shoots of Nicotiana rustica L. regreened, especially when treated with cytokinin. Regreening caused an increase in leaf thickness, due to cell expansion. Senescent leaf plastids (gerontoplasts) were smaller than green chloroplasts, with degenerated membrane systems and stroma, and larger plastoglobuli. At advanced senescence, micrographs showed disintegrating gerontoplasts, reduced numbers of plastids were counted, and regreening became variable. The redevelopment of grana and stroma in regreening plastids was accelerated by cytokinin. All plastids in regreening leaves were identifiable as redifferentiating gerontoplasts because of their content of plastoglobuli and starch. Immunogold labelling showed significant association of POR with etioplasts in cotyledons, but with mature plastids in regreening leaves. No proplastids or dividing chloroplasts were observed in regreening leaves. Plastids numbers declined during senescence and did not increase again during regreening. It is concluded that the chloroplasts of regreening leaves arose by redifferentiation of gerontoplasts.Keywords: Chloroplasts, cytokinin, Nicotiana, senescence, regreening.      

Comparative ultrastructural properties of pallisade tissue and spongy tissue chloroplasts from normal and mutant leaves of Pisum sativum L.*

Abstract. The ultrastructure of chloroplasts from palisade and spongy tissue was studied in order to analyse the adaptation of chloroplasts to the light gradient within the bifacial leaves of pea. Chloroplasts of two nuclear gene mutants of Pisum sativum (chlorotica-29 and chlorophyll b-less 130A), grown under normal light conditions, were compared with the wild type (WT) garden-pea cv. ‘Dippes Gelbe Viktoria’. The differentiation of the thylakoid membrane system of plastids from normal pea leaves exhibited nearly the same degree of grana formation in palisade and in spongy tissue. Using morphometrical measurements, only a slight increase in grana stacking capacity was found in chloroplasts of spongy tissue. In contrast, chloroplasts of mutant leaves differed in grana development in palisade and spongy tissue, respectively. Their thylakoid systems appeared to be disorganized and not developed as much as in chloroplasts from normal pea leaves. Grana contained fewer lamellae per granum, the number of grana per chloroplast section was reduced and the length of appressed thylakoid regions was decreased. Nevertheless, chloroplasts of the mutants were always differentiated into grana and stroma thylakoids. The structural changes observed and the reduction of the total chlorophyll content correlated with alterations in the polypeptide composition of thylakoid membrane preparations from mutant chloroplasts. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polypeptide bands with a relative molecular mass of 27 and 26 kilodalton (kD) were markedly reduced in mutant chloroplasts. These two polypeptides represented the major apoproteins of the light harvesting chlorophyll a/b complex from photosystem II (LHC-II) as inferred from a comparison with the electrophoretic mobility of polypeptides isolated from the LHC-II.     

RECONSTRUCTIONS OF THE GRANA-FRETWORK SYSTEM OF A CHLOROPLAST

The grana-fretwork system was reconstructed from serial sections representing 0.5 μm slice, in profile view, from the midregion of a chloroplast of Nicotiana tabacum. Reconstructions show grana in multiple strata. The fretwork integrates the grana in all three dimensions. Large grana are visualized as complex structures consisting of two or more eccentrically stacked solids that vary in height and diameter. The close spacing and overlapping positions of grana in the plastid explain why phase microscopy cannot be used to demonstrate adequately the numerous strata of grana within a chloroplast. The grana are not far enough apart to allow for successful optical sectioning of a plastid in face view. In profile view no distinct grana can be resolved because the grana-fretwork system has the aspect of a “honeycomb.” Thus, observations with a light microscope are not adequate to determine the arrangement of grana in a chloroplast, and the recently-proposed model showing all grana of a plastid to be arranged in a single spiral ribbon must be rejected as incompatible with properties of real plastids.     

Silencing of NbCEP1 encoding a chloroplast envelope protein containing 15 leucine-rich-repeats disrupts chloroplast biogenesis in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

We characterized the physiological functions of Nicotiana benthamiana Chloroplast Envelope Protein 1 (NbCEP1) in Nicotiana benthamiana. NbCEP1 contains a chloroplast transit peptide and a single transmembrane domain at the N terminus, and most of its protein coding region is comprised of 15 leucine-rich-repeats (LRRs). The NbCEP1 gene is expressed in both aerial and underground plant tissues, and is induced by light. A GFP fusion protein of full length NbCEP1 was targeted to the chloroplast envelope and co-localized with OEP7:RFP, a marker protein for the chloroplast envelope. A fusion protein consisting of GFP and the NbCEP1 transit peptide mainly localized in the chloroplast stroma. Reduction of NbCEP1 expression by virus-induced gene silencing resulted in a leaf yellowing phenotype without much affecting overall plant growth. At the cellular level, depletion of NbCEP1 severely influenced chloroplast development, reducing both the number and size of the chloroplasts. Interestingly, mitochondrial development was also impaired, possibly an indirect effect of chloroplast ablation. A deficiency in NbCEP1 activity decreased the chlorophyll and carotenoid levels. Our results suggest that NbCEP1 plays a critical function, possibly through protein-protein interactions mediated by its LRRs, in chloroplast development in N. benthamiana.     

THE GREENING OF ETIOLATED BEAN LEAVES AND THE DEVELOPMENT OF CHLOROPLAST FINE STRUCTURE IN ABSENCE OF PHOTOSYNTHESIS

CMU inhibits oxygen evolution in greening etiolated bean leaves.In the presence of this compound chlorophyll content is reducedand fine structure development of the chloroplasts is markedlyaffected. The number of grana per chloroplast is reduced butthe grana are larger and contain more thylakoids than the granain chloroplasts of the greening control leaves. Sucrose reversesthe effect of CMU on pigment content and fine structure developmentof chloroplasts. (Received September 14, 1965; )     

小麦黄化突变体叶绿体超微结构研究

利用透射电镜对小麦自然黄化突变体及其突变亲本(西农1718)叶片细胞叶绿体的数目、形态及超微结构进行比较分析。结果发现:(1)3种不同黄化程度突变体的叶绿体分布、数目、形状及大小与突变亲本无明显差异;(2)突变体叶绿素含量为野生型58%的黄绿植株与其突变亲本叶绿体超微结构无明显差异,基质类囊体与基粒类囊体高度分化,基粒数目以及基粒片层数目较多;(3)突变体金黄和绿黄植株的叶绿素含量分别为野生型的17%、24%,其叶绿体超微结构与突变亲本明显不同,突变体的叶绿体发育存在明显缺陷,其中突变体金黄植株的叶绿体内无基粒、基质片层清晰可见,有淀粉粒,嗜锇颗粒较多,而突变体绿黄植株的叶绿体内有基粒,但明显少于突变亲本,且基粒片层较少,基质类囊体较发达。结果表明该黄化突变体叶绿体超微结构的改变,是由于叶绿素含量降低造成,推测,该黄化突变是由于叶绿素合成受阻导致的。     

Stromal low temperature compartment derived from the inner membrane of the chloroplast envelope

Leaf discs of four dicotyledonous species, when incubated at temperatures of 4 to 18°C (optimum at 12°C) for 30 or 60 minutes, responded by accumulations of membranes in the chloroplast stroma in the space between the inner membrane of the envelope and the thylakoids. The accumulated membranes, here referred to as the low temperature compartment, were frequently continuous with the envelope membrane and exhibited kinetics of formation consistent with a derivation from the envelope. Results were similar for expanding leaves of garden pea (Pisum sativum), soybean (Glycine max), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum). We suggest that the stromal low temperature compartment may be analogous to the compartment induced to form between the transitional endoplasmic reticulum and the Golgi apparatus at low temperatures. The findings provide evidence for the possibility of a vesicular transfer of membrane constituents between the inner membrane of the chloroplast envelope and the thylakoids of mature chloroplasts in expanding leaves.     

遮荫对南美蟛蜞菊、蟛蜞菊及其自然杂交种叶片显微结构及叶绿体超微结构的影响

利用JSM-6360LV型扫描电镜和JEM-1010型透射电镜,观察了南美蟛蜞菊、蟛蜞菊及其自然杂交种新近成熟和老熟叶片的解剖结构及叶绿体超微结构。结果表明:遮荫后该杂交种与其亲本新近成熟叶片均表现为上下表皮气孔密度、叶片总厚度及上下表皮厚度、栅栏组织、海绵组织厚度减小,叶绿体肿胀变形,基粒片层垛叠程度增加,淀粉粒增多变大;遮荫后杂交种老熟叶片总厚度及上表皮、栅栏组织、海绵组织厚度增加,入侵种的下表皮厚度及本地种的上表皮厚度增加,叶绿体超微结构在遮荫后均出现严重损伤,基粒片层类囊体结构边缘溶解等。说明三种蟛蜞菊属物种及各物种不同叶龄叶片对弱光条件的响应存在差异;杂交种叶片显微及超微结构在不同光照下的变化介于亲本之间,对遮荫环境能较好适应。     

青藏高原萹蓄、车前叶绿体超微结构研究

对生长在青藏高原两个海拔高度的蓄和车前叶绿体进行超微观察表明。1.高海拔地区蓄的叶绿体发生变形,叶绿体的长度缩短、厚度增加,被膜模糊,而车前叶绿体的形态变化不大。2.高海拔地区的两种植物叶绿体的基粒片层和基质片层都呈现肿胀现象,尤以蓄为显著。3．高海拔地区的两种植物叶绿体中基粒片层的叠垛程度均比低海拔地区的高。以上特征是青藏高原特殊的高寒生态条件长期影响的结果。