Thylakoid System Research Articles

AN ALBINO MUTANT IN PLANTAGO INSULARIS REQUIRING THIAMINE PYROPHOSPHATE—EFFECTS OF TPP ON CHLOROPLAST ULTRASTRUCTURE

SUMMARYThe responses of normal (al+/al+) and albino mutant (aljal) seedlings of Plantago insularis to light and thiamine pyrophosphate (TPP) were studied by transmission electron microscopy.Plastids in seedlings of normal and albino genotypes grown in darkness had the structure of etioplasts with a thylakoid plexus (prolamellar body) from which extend thylakoids. Some of the thylakoids were often associated into parallel or concentric sheets. In the mutant, the thylakoid sheets were generally shorter, and some were vesiculated. Exposure of normal plants to light resulted in the transformation of the etioplasts to chloroplasts, with the loss of the thylakoid plexus and the formation of typical grana and an intergranal thylakoid system, plus a few globules in the stroma. Exposure of the albino mutant to light caused the loss of the thylakoid plexus and the formation of highly ordered, concentrically associated thylakoid sheet systems, but no grana. After 24 h of illumination, the plastids of the albino mutant showed evidence of thylakoid degeneration and accumulation of globules and starch. These changes are interpreted as a consequence of very low TPP in the mutant and as evidence for the role of TPP in the degradation of glucose through the Krebs cycle in the normal genotype.The application of TPP to the albino mutant resulted in the formation of essentially normal chloroplasts. The ultrastructural observations support the view that TPP deficiency does not handicap the mutant in protein synthesis until after exposure to light. TPP and light are essential for the formation of normal grana and TPP is in some way involved in the pathways for pigment and protein synthesis in chloroplasts.

Seasonal changes in structure and function of spruce chloroplasts

Seasonal changes of ultrastructure were studied by electron microscopy and by determining the chlorophyll and starch content of the plastids. Young plastids of spruce (Picea abies (L.) Karst.) first function as amyloplasts which store reserve material for the growth of the young needles. Then they develop a normal thylakoid system and produce assimilation starch during the day. In autumn, starch synthesis ceases and the plastids group together. In winter they swell and their membrane system becomes disorganized and reduced. With increasing temperatures in spring the chloroplasts recover, but then they accumulate large amounts of starch, which is not broken down during the night or even during a dark period of several days. As in the previous year they now function as amyloplasts providing reserve material for the new shoot. In summer these plastids are again converted into typical chloroplasts. The same seasonal changes of structure and function could be observed in chloroplasts from 2- or 3-year old needles. Thus these changes represent cyclic processes, which repeat each year. Features of slow aging are superimposed on to these cycles.

Relative thermostability of the chloroplast envelope

Intact isolated chloroplasts from leaves of Spinacia oleracea L. were subjected to heat treatment. After heating, the integrity of the chloroplast envelopes and the activities of various light-dependent chloroplast reactions were tested. The integrity of the chloroplast envelopes, as judged from rates of ferricyanide reduction, enzyme compartmentation and visual appearance of the chloroplasts in the light microscope with phase optics, was affected much less by heat stress than the photochemical reactions of thylakoids. This indicates a comparatively high thermostability of the chloroplast envelope membranes. It is also evidence of a differential thermostability of different biomembranes. Photophosphorylation was highly susceptible to thermal stress. Heat treatment that partly inactivated phosphorylation stimulated light-dependent quenching of 9-aminoacridine fluorescence, which served as an indicator of proton transfer from stroma to thylakoids in intact chloroplasts. Drastic changes in the characteristics of chlorophyll a fluorescence emission caused by heating were probably due to structural alterations of the thylakoid system.

Effects of Green Light on the Chloroplasts of Spinach Leaf Discs

The development and photosynthetic capacity of chloroplasts formed in green light in cultured spinach leaf discs has been studied. At intensities of 4 to 6 mW cm-2 green light stimulates chloroplast replication to about the same extent as white, blue, and red light. However, practically no chloroplast replication occurs in discs grown in low intensity green or white light but considerable chloroplast growth takes place. Ultrastructural studies have shown that these chloroplasts, which can be two to five times the area of control plastids (high intensity white light), have an essentially normal thylakoid system. Double isotope labelling experi ments have established that the synthesis of chloroplast ribosomal-RNA is similar in controls and in discs grown in low intensity green or white light. On a per unit chlorophyll basis the CO2 fixation rate of spinach discs grown in low intensity green (or white) light saturates with increasing light intensity or increasing CO2 concentration at values well below control discs. In this respect their photosynthetic characteristics bear a similarity to those of shade plants.

Electrochemical Phenomena Associated with the “Light Reaction” in Chloroplast Thylakoids

Some observations are reported resulting from imposed “null potential” conditions on untreated thylakoid suspensions during successive periods of illumination and after chemical and physical modifications that affect “normal” behaviour. Comparison of normal coulombic outputs puts with those obtained from thylakoids in the presence of several inhibitors of electron flow from water to the reducing side of photo-system II, and with an inhibitor of photosystem I imply that the electrons detected are derived from the decomposition of water and are transferred to the electrode from the reducing side of photosystem I. Imposing more positive or less positive conditions of potentiostatic means prior to illumination at “null potential” resulted in signifant alterations of the parameters being measured. Similar abnormal effects were noted when the thylakoid system was examined under oxygen-saturated and anaerobic conditions. The results suggest that under circumstances wherein more positive conditions exists, the photosensitisers might in their more oxidised state, being hyperactivated receptors, increase the rate of water decomposition, resulting in concurrent increase in electron release. Conditions less positive than the “null state” result in an opposite effect.

Mobility of chloroplast coupling factor 1 (CF1) at the thylakoid surface as revealed by freeze-etching after antibody labelling.

Abstract Freeze-Etching Freeze-fracturing and 60 sec deep-etching of isolated chloroplast thylakoid systems exposed large areas of the outer surface (matrix side) of the thylakoids. If the thylakoid systems were first treated with antisera against chloroplast coupling factor 1 (CF1), the 14 nm particles at the outer surface appeared aggregated. Between clusters these particles were absent. Since there is no change in the number of particles/area after treatment with antibodies, it is concluded that the 14 nm particles are mobile within the surface of the thylakoid. The antisera contained only anti­bodies against CF1 ; therefore the 14 nm particles at the outer surface are identified to be CF1 . The implication of a mobile ATP-synthetase (CF1) for the mechanisms of photophosphorylation is discussed.

Plastiden-metamorphose und chromoplasten bei Chrysosplenium

Summary 1. The leaves of the Golden Saxifrage, Chrysosplenium alternifolium and Chr. oppositifolium , are deeply green, whereas the bracts and the small leaflets of the calyx (a corolla is lacking) appear (greenish-)yellow during flowering time. Correspondingly, leaf cells contain chloroplasts, and bract cells chloro-chromoplasts. The sepals contain chromoplasts, especially in their epidermal cells. 2. The sepals exhibit remarkable changes in color. They are green when the flower buds break open, and yellow during anthesis. During seed development they enlarge and turn green again. A reversible plastid metamorphosis is responsible for these color changes. 3. The fine structure of leaf and calyx plastids, and metamorphosis of plastids in calyx leaflets in particular, has been investigated (mainly in Chr. alternifolium) by conventional electron microscopy. The pictures have been analyzed by morphometric methods (for results, compare fig. 6). 4. Mesophyll chloroplasts of green leaves are uneven-shaped. The numerous grana are oriented in different planes and comprise up to 30 thylakoids in one granum (figs. 11 a, b). Starch grains are always present. Osmiophilic plastoglobules are numerous but rather small (Fig. 9). 5. The sepals contain, in every state of their development, typical chromoplasts in their upper epidermis. These chromoplasts are small and flat (figs 3, 4 and 8). They contain, in a central layer, a few thylakoids and rudimentary grana with but a few thylakoids per granum (fig. 7, see also table 1). The periphery of the chromoplasts is filled with large-size, osmiophilic plastoglobules. They are mostly transformed into polyhedral crystals in the open flower (fig. 4). After anthesis, these carotenoid-containing crystals partly enlarge. Simultaneously, new spherical and often less osmiophilic plastoglobules are formed in a more central region of the chromoplasts (fig. 8). On the whole, metamorphosis of plastids is “monotropic” in the upper sepal epidermis. 6. Macroscopic color changes of the sepals are due to a reversible chloroplast-to-chromoplast transformation in the mesophyll. At the outset of anthesis, the plastids of these cells are typical lens-shaped, starch-containing chloroplasts (fig. 2). During anthesis, chromoplasts differentiate by enlargement of plastoglobules and concomitant reduction of the thylakoid and grana system as well as of stroma matrix material and starch (fig. 5). These chromoplasts are considerably smaller than the chloroplasts from which they originate (fig. 1). Later, during seed formation, these mesophyll chromoplasts of the sepals revert to chloroplasts by reforming larger grana und starch grains, and by reducing the size of the chlotenoid-carrying plastoglobules. Hence, these plastids behave quite differently from the epidermal plastids of the very same leaflet, and thus present a good example of a “non-monotropic”, reversible transformation. 7. Regarding the other components of sepal cells, no conspicuous changes have been observed. 8. These results are discussed in relation to some data of the chromoplast literature, and a contemporary classification of different chromoplast fine structure types is given.

Zur physiologischen Bedeutung von Chlorophyll b

In order to investigate the function of chlorophyll b three mutants without chlorophyll b were compared with the original form of Hordeum vulgare cv. Donaria. They show decreases in dry matter production, in chlorophyll a content, and in grain yield. Their rate of photosynthesis was lowered to a small extent, but not as the consequence of elevated photorespiration. The HILL reaction with ferricyanid was higher at light saturation than that of the parent form Donaria when related to chlorophyll a; if calculated per chloroplast the mutant 3613 showed equal HILL reaction activity. The mutants formed well developed thylakoid systems and exerted non-cyclic photophosphorylation. The mutants were able to form starch in the chloroplasts of the parenchym cells.

The Fine Structure of Avocado Plastids

Ultrastructural studies of both young and harvest-ripe avocado fruits have established that the skin and outer green layers of flesh contain chloroplasts with an extensive thylakoid system. Etioplasts occur in the yellow flesh adjacent to the stone. The pale-green flesh contains plastids, intermediate between chloroplasts and etioplasts, which have prominent prolamellar bodies from which radiate grana. When segments of both the yellow and pale-green flesh of mature fruit (7 cm diam.) are cultured in the light their prolamellar bodies do not disperse although there is a change in their crystallinity. The palegreen tissues of immature (4 mm and 2 cm diam.) fruits also contain etioplasts but on culturing these differentiate into chloroplasts. Both chlorophyll content and the ratio of chlorophyll a to b varied in the different tissues of young and mature fruits.

LIGHT AND ELECTRON MICROSCOPE OBSERVATIONS ON ALGAL LYSIS BY BACTERIUM CP‐I

SummaryBacterium CP‐I isolated from Scottish waters is an aerobic prokaryote with a high guanine: cyto‐sine ratio (68%), which causes lysis of a variety of blue‐green algae. When suspensions of algae and bacteria are mixed the bacteria can be seen to glide towards the algae where they become attached end‐on round the algal cells. There is evidence that the bacteria produce substances at the point of contact with the algae which are necessary for algal lysis to occur. Lysis of vegetative cells may occur within 30 minutes of the bacteria becoming attached. Heterocysts do not lyse in this way although their intracellular contents may eventually degenerate. When vegetative cells rupture, the protoplasmic contents, including gas vacuoles when present, are released and empty‐looking ‘ghosts’ which retain the original cell shape may remain for a time although these too eventually break down. There is some evidence that the mode of filament lysis may be random, intercalary or terminal.At the ultrastructural level the initial effect of CP‐I is to cause lysis of the L2 layer of the algal cell wall and this effect is similar to that obtained using lysozyme. This is followed by rupture or disintegration of the other cell wall layers, and ‘scroll’‐like structures, either of coiled up cell wall material (Microcystis) or of the plasmalemma (Nostoc) may be found scattered throughout the preparations of lysed cells. In fully lysed cells there is a loss of polyphosphate bodies, structured granules, gas vacuoles, and most other cellular inclusions. Released gas vesicles can be collected from the surface of the solution of lysed cells, and there is evidence that these vesicles may fracture easily near the conical ends. In fully lysed cells, the thylakoid membranes remain, often with wide lumens between. This thylakoidal system is contorted and branched, ramifies through the cell and shows constrictions of the membrane system at intervals. Continuity between the plasmalemma and the thylakoidal system can be seen clearly, Lipid droplets which are resistant to CP‐I treatment are found in the lumen of the thylakoidal system. Heterocysts are not as markedly affected by CP‐I as are vegetative cells, but eventually heterocyst disorganization also occurs. The use of CP‐I provides a technique for preparing material for the study of algal membranes and gas vesicles.

The inhibition of kohlrabi chloroplast degeneration by kinetin.

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 (Denniset al. 1967), which suggests that the plastid degeneration model may be specific for speciesBrassica oleracea L.

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”). 1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride. 2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage. 3. 3. In intact leaves under strong illumination with red light in CO 2-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage. 4. 4. Under nitrogen, CO 2 caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO 2 levels fluorescence was increased and shrinkage decreased. 5. 5. In the presence of CO 2, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air. 6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

The effect of 3-Indolylacetic acid on the differentiation of plastids in callus culture ofCichorium intybus L.

The effect of 3-indolylacetic acid (IAA) on structure of plastids in cells of callus developing on the phloem explants of chicory roots was investigated. In the absence of IAA the proplastids in the initial expiant developed into typical chloroplasts. The presence of increasing IAA concentrations in the medium resulted in a gradual reduction of the thylakoid system accompanied by an increasing starch content of the plastids. Depending on the IAA concentration used, various types of plastids from typical chloroplasts to typical amyloplasts were found. A possible relationship between auxins and sugar metabolism is indicated.

Maternal inheritance, cytology and macromolecular composition of defective chloroplasts in a variegated mutant of Nicotiana tabacum

By phase microscopy of living cells the cause of a maternally-inherited variegated, spontaneous mutation of Nicotiana tabacum L. cv. Turkish Samsun was shown to be the presence of defective chloroplasts. These were intermingled with normal chloroplasts in some of the cells of the mesophyll tissue. In young, expanding leaves, the defective chloroplasts contain traces of chlorophylls a and b in the same ratio as found in normal chloroplasts, but only one-thirtieth of the quantity. As the defective chloroplasts mature, the green pigments disappear. The defective chloroplasts thus appear to be greatly deficient in thylakoid membranes. From their dynamic changes in shape, the defective chloroplasts appear to consist almost entirely of mobile phase, the structure which surrounds the thylakoid system of membranes of normal chloroplasts of higher plants. Consistent with this idea, two constitutents located in the mobile phase of normal chloroplasts-70S ribosomes and Fraction I protein-were detected in defective chloroplasts. The Fraction I protein was unchanged in specific ribulose diphosphate carboxylase activity from enzyme isolated from normal chloroplasts. Speculations are presented that the mutation in chloroplast DNA responsible for the formation of defective chloroplasts cannot be attributed to cistrons coding for the protein of Photosystem II, chloroplast ribosomal RNA or proteins, Fraction I protein, or the DNA-dependent RNA polymerase of chloroplasts.

Ultrastructural Changes in Leaf Tissues of Populusdeltoides spp. angulata Ait. Infected with Poplar Mosaic Virus

Ultrastructural observations on the foliar cells of Populusdeltoides spp. angulata Ait. infected by poplar mosaic virus revealed a high content of virus particles in the cytoplasm of most but not all young cells from chlorotic areas. Mature cells from necrotic areas contained far fewer virions while in symptomless green portions no virions were observed. Virus particles were not found in mature sieve elements or in mature nonliving xylem cells.Changes in the cytoplasm and in chloroplasts were followed. Healthy, mature chloroplasts contained well developed thylakoid systems. In infected cells, a disintegrative process at necrotization resulted in the dissolution of the thylakoid system into individual electron dense sacs which were released when the chloroplast membrane dissolved. Cytoplasmic changes were characterized by increased vacuolation. Cisternae of the endoplasmic reticulum and vesiculate bodies were also more numerous. Necrotization was accompanied by dissolution of the cell organelles.

Plants under Climatic Stress

Mesophyll chloroplasts of the C(4)-pathway grasses Sorghum and Paspalum and of the C(3)-pathway legume soybean undergo ultrastructural changes under moderate light intensities (170 w.m(-2), 400-700 nanometers) at a tme when photosynthesis is much reduced by low temperature (10 C). The pattern of ultrastructural change was similar in these species, despite some differences in the initial sites of low temperature action on photosynthesis and differences in their mechanisms of CO(2) fixation. Starch grains in the chloroplasts rapidly reduce in size when chilling stress is applied. At or before the time starch grains completely disappear the membranes of the individual stromal thylakoids close together, reducing the intraspace between them while the chloroplast as a whole begins to swell. Extensive granal stacking appears to hold the thylakoids in position for some time, causing initial swelling to occur in the zone of the peripheral reticulum, when present. At more advanced stages of swelling the thylakoid system unravels while the thylakoid intraspaces dilate markedly. Initial thylakoid intraspace contraction is tentatively ascribed to an increase in the transmembrane hydrogen ion gradient causing movement of cations and undissociated organic acids from the thylakoid intraspace to the stroma. Chloroplast swelling may be caused by a hold-up of some osmotically active photosynthetic product in the chloroplast stroma. After granal unraveling and redilation of the thylakoid intraspaces, chloroplasts appear similar to those isolated in low salt hypotonic media. At the initial stages of stress-induced ultrastructural change, a marked gradient in degree of chloroplast swelling is seen within and between cells, being most pronounced near the surface of the leaf directly exposed to light.

ULTRASTRUCTURAL CHANGES ASSOCIATED WITH UTILIZATION OF METABOLITE RESERVES AND TRICHOME DIFFERENTIATION IN THE PROTOCORM OF VANDA

Certain aspects of protocorm development in Vanda were examined ultrastructurally. The parenchymal cells of the protocorm accumulate substantial quantities of lipid, protein, and carbohydrate reserves which disappear gradually with the senescence of the parenchymatous region. The proteinaceous reserves appear initially as discrete bodies which become intimately associated with clusters of small tubules. The tubules eventually disperse throughout the cytoplasm and disappear following depletion of the protein bodies. The lipid reserves also appear as discrete bodies and are associated with an electron dense, laminated inclusion which appears to increase in size with the disappearance of the lipid bodies. While plastids in the meristematic cells differentiate a well‐developed thylakoid system and contain little starch, those of the parenchymal cells contain large starch grains and numerous osmiophilic droplets and develop meager thylakoid systems. Membrane‐bound crystalline structures of hexagonal and rhomboid cross section occur frequently in the cytoplasm of senescent parenchyma cells. Trichome initials, which differentiate from the epidermis, contain few conventional organelles and exhibit numerous membrane‐bound structures containing many small crystalline inclusions. Numerous vesicles accumulate at the tips of the trichomes in spaces between the cell wall and the plasmalemma.

On a water soluble factor neutralizing antibodies against the primary acceptor in photosynthetic electron transport of chloroplasts

Rabbits immunized against the thylakoid system of chloroplasts form two different antibodies against the reducing site of photosystem I. One inhibits photosynthetic NADP(+) and ferredoxin dependent cytochrome c reduction, but not photosynthetic anthraquinone reduction by isolated spinach chloroplasts. The other inhibits all three reductions. Both do not inhibit a Hill reaction with ferricyanide. The presumed antigen moiety against these antibodies was isolated as a water soluble factor released from lyophilized chloroplasts after treatment with diethylether. The heat stable, nondialysable factor has absorption peaks at 262 and 315 mμ. It is autoxidizable and has the property of a cytochrome c reducing substance. The factor neutralizes the antibody inhibition of photosynthetic anthraquinone and ferredoxin reduction. It is proposed that the soluble factor is the prosthetic group of the primary acceptor of photosystem I.

Phylogenetic Transitions in the Chloroplasts of the Anthocerotales. I. The Number and Ultrastructure of the Mature Plastids

Representatives of three genera of anthooerotes were examined: Phaeoceros, Notothylas, and Megaceros. Species of the first two genera were found to exemplify the typical anthocerote plastid condition. This condition is characterized by the presence in each cell of the gametophyte of only a single large chloroplast containing a “multiple” pyrenoid. The genus Megaceros, however, proved to be quite different. In two species of Megaceros the pyrenoid was observed to be composed of a highly subdivided thylakoid system of even greater complexity than the “multiple” pyrenoids of Phaeoceros. In another species only an indistinct “pyrenoid-like” area was noted while in a fourth species no evidence was found for any internal differentiation. Associated with these changes in plastid structure there are corresponding alterations in the number and the size of the chloroplasts. Together they indicate an evolutionary trend away from a primitive, algal-like condition to a more advanced land plant form.

PHYLOGENETIC TRANSITIONS IN THE CHLOROPLASTS OF THE ANTHOCEROTALES I. THE NUMBER AND ULTRASTRUCTURE OF THE MATURE PLASTIDS

Representatives of three genera of anthooerotes were examined: Phaeoceros, Notothylas, and Megaceros. Species of the first two genera were found to exemplify the typical anthocerote plastid condition. This condition is characterized by the presence in each cell of the gametophyte of only a single large chloroplast containing a “multiple” pyrenoid. The genus Megaceros, however, proved to be quite different. In two species of Megaceros the pyrenoid was observed to be composed of a highly subdivided thylakoid system of even greater complexity than the “multiple” pyrenoids of Phaeoceros. In another species only an indistinct “pyrenoid‐like” area was noted while in a fourth species no evidence was found for any internal differentiation. Associated with these changes in plastid structure there are corresponding alterations in the number and the size of the chloroplasts. Together they indicate an evolutionary trend away from a primitive, algal‐like condition to a more advanced land plant form.