Chloroplast Formation Research Articles

Heat-induced changes in dry weight, leaf size and number of chloroplasts per cell in maize and cowpea shoots

SUMMARYWhen etiolated seedlings of maize and cowpea were subjected to a heat stress of 45 °C for 8 h and subsequently illuminated, there was a retardation in the rates of leaf elongation and chloroplast formation, but the rate of increase in dry weight was not affected. However, a heat stress of 45 °C for 16 h decreased all the three rates measured, the effects being more pronounced with the longer duration of stress in the case of leaf elongation and chloroplast formation. The cowpea seedlings were more sensitive to heat than the maize seedlings. The decreased yield (dry weight) in the 16 h treatment was due, at least partly, to the heat-induced retardation of the growth of the photosynthetic apparatus.

A Light-induced Structural Change in Chloroplasts of Chinese Cabbage Cells Infected with Turnip Yellow Mosaic Virus

SUMMARY When protoplasts are prepared from Chinese cabbage leaves 5 to 20 days after inoculation with turnip yellow mosaic virus (TYMV) a variable proportion of the cells possess the rounded and clumped chloroplasts typical of infection with this virus while others appear normal. On exposure to light normal chloroplasts remain unchanged while chloroplasts in diseased cells undergo a striking structural transformation. A large clear vesicle appears in the chloroplast, the chlorophyll-bearing structures being confined to a crescent-shaped fraction of the chloroplast volume. This ‘sickling’ effect occurs only in the light. Red and blue light are equally effective. There is a lag period of about 4 h in the light before protoplasts with sickled chloroplasts begin to appear. The proportion of protoplasts showing sickled chloroplasts after 24 h exposure increases with increasing light intensity up to about 2000 lux. The production of sickled cells is partially suppressed when glucose, fructose, sucrose, or certain other sugars are added to the incubation medium. The electron transport inhibitor DCMU completely suppresses the formation of sickled chloroplasts at a concentration of 10−6 m.

Chloroplast replication and chloroplast DNA synthesis in spinach leaves

Cultured leaf disks from young spinach leaves have been used to study chloroplast DNA synthesis in relation to chloroplast growth and replication. Autoradiographs prepared from thin sections show that labelling throughout disks is uniform. We consider that cultured leaf disks serve as a model system for studies of cell expansion and chloroplast replication during the development of spinach leaf cells. Leaf disks from young spinach leaves were incubated continuously for 5 days in a medium containing [ 3 H]thymidine. They were harvested daily and light microscope autoradiographs of separated cells prepared. In the expanding cells of disks all chloroplasts divide and the synthesis of chloroplast DNA as measured from grain counts was correlated with the rate of chloroplast formation. A doubling of chloroplast numbers was associated with a doubling of chloroplast silver grains, which is consistent with a duplication of chloroplast DNA during the chloroplast division cycle. When disks were grown in green light, chloroplast replication but not chloroplast growth ceased in 3–4 days. The large chloroplasts of disks grown in green light appeared locked in a dumb-bell shaped configuration. Light microscope autoradiographs have established that these chloroplasts can synthesize DNA. These chloroplasts divided when the disks were exposed to high intensity white light and again it appears that all chloroplasts in the one cell divide. The chloroplasts of disks grown in green light for periods up to 9 days became uneven in size although all were dumb-bell shaped. We suggest that the size differences represent ploidy classes. Chloroplast replication in dicotyledons has been discussed in relation to the division and growth of leaf cells.

Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.

Formation of prolamellar bodies without correlative accumulation of protochlorophyllide or chlorophyllide in pine cotyledons

Chloroplast formation and accumulation of chlorophyllous pigments were studied in pine cotyledons ( Pinus jeffreyi) during germination in complete darkness or under continuous light. During the first days of germination (in the light and in the dark), cotyledons grew inside the seed; at this moment, no membranes were visible in proplastids and no chlorophyllous pigments could be detected. When the radicle broke the seed integuments, the cotyledons had developed green plastids. These contained crystalline prolamellar bodies as well as lamellae and grana. The same plastid structure was found in fully expanded cotyledons from dark-brown pine seedlings. Under continuous light, even when cotyledons emerged from the seed integuments, the plastids still contained prolamellar bodies. The prolamellar bodies remained for several days but gradually decreased and finally disappeared. Analysis of pigment content could not establish a correlation between the formation of prolamellar bodies and the accumulation of protochlorophyllide a or/and chlorophyllide a.

Effects of nalidixic acid, chloramphenicol, cycloheximide, and anisomycin on structure and development of plastids and mitochondria in greening Euglena gracilis

The substructure of plastids and mitochondria and the alterations caused by the addition of antibiotics were investigated during light-induced proplastid-to-chloroplast transformation in Euglena gracilis. The organisms were grown in presence of the inhibitors up to 3 days (5 generations). Both 40 μg/ml nalidixic acid and 1–1.5 mg/ml chloramphenicol prevent the formation of chloroplasts of normal size and structure by blocking development during early stages. Under our conditions 2 to 5 straight thylakoids are formed beside 1 to 2 girdle-like thylakoids. The former rarely fuse into bands. Non-crystalline prolamellar bodies of considerable size are formed at the distended ends of the plastids in the presence of both drugs. Chloramphenicol also influences mitochondrial size, shape and internal structure. Giant mitochondria can be observed. Nalidixic acid does not change the size and shape of mitochondria, but the matrix frequently appears highly osmiophilic. Cycloheximide in sublethal doses (2–5 μg/ml) or 50 μg/ml anisomycin inhibits plastid development only in the early period after addition. In later culture periods chloroplasts are found enlarged in size with an increased number of thylakoids and bands per organelle. Insertions of new bands are noted at the inner membrane of the chloroplast envelope. The electronmicroscopic observations agree with the results of chloroplast-specific biochemical activities such as light-induced increase in chlorophyll synthesis and of two chloroplast-bound enzyme activities. The results are discussed with respect to metabolic and biogenetic correlations between the two types of organelles in E. gracilis cells.

Appearance of Three Chloroplast Isoenzymes in Dark-grown Pea Plants and Pea Seeds

Activity peaks characteristic of the chloroplastic Calvin cycle enzymes triose-phosphate isomerase, ribose 5-phosphate isomerase, and fructose 1,6-diphosphate aldolase are found in isoelectric focusing patterns of dark-grown pea (Pisum sativum) seedlings and seeds. Apparently, in this higher plant these three chloroplastic isoenzymes can be formed in the absence of light and of chloroplast formation.

Identification of a mutation in chloroplast DNA correlated with formation of defective chloroplasts in a variegated mutant of Nicotiana tabacum

As in wild-type Nicotiana tabacum L., two satellite DNAs having densities of 1.700 and 1.705 g cm(-3) in CsCl were identified in the organelle fraction of homogenates made from variegated leaves of a cytoplasmic mutant of N. tabacum. As the proportion of white to green tissue increased a great reduction in the 1.700 chloroplast DNA occurred correlated with a concomitant reduction in the total number of defective and normal chloroplasts per cell. At the same time, there was an absolute increase in the 1.705 satellite DNA. Separation of the two satellite DNAs was achieved by one cycle of purification on NaI gradients. When the 1.700 chloroplast DNAs from white and from green tissue of variegated leaves were compared, identical properties were found by the conventional buoyant density, T m and renaturation kinetics measurements. However, using a specially constructed difference melting system, the 1.700 DNA from defective chloroplasts was shown to have an approximately 1% higher GC composition than the DNA from normal chloroplasts. Also, by renaturation of a mixture of alkali denatured normal and defective chloroplast DNAs and subsequent spreading in formamide for electron microscopy, internal regions of mismatching were observed. The nonhomologous region corresponded to about 500-1000 base pairs. No differences in composition of the 1.705 satellite DNA derived from white or green tissues were detected either by difference melting or formation of heteroduplexes.

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.

The development, inheritance, and origin of the plastid in Euglena.

This chapter describes the development, inheritance, and origin of the plastid in Euglena . The nucleus, chloroplast, and mitochondrion are cellular organelles having double membranes and the ability to divide. A significant proportion of the energy and small molecules required for plastid development comes from outside the chloroplast and some of it from the mitochondrion. The information for constructing plastid proteins originates in the plastid and nuclear DNAs. The translation of nuclear information occurs on cytoplasmic ribosomes while the translation of plastid information occurs on plastid ribosomes. Cytoplasmic contributions to plastid formation begin earlier than plastid contributions during light-induced chloroplast formation, and the lag period during the first 12 hours is devoted to cytoplasmic events preceding the developmental changes in the plastid. The plastid in Euglena is prokaryotic in structure and composition, and the plastid originated from the endosymbiotic invasion of an organism that is similar to a primitive blue-green alga or the contemporary proplastid.

Induction of photosystem II activity in flashed leaves

The formation of photosynthetically functioning, normal chloroplasts in higher plants by exposure of etiolated leaves to light is well documented: morphologically [9] , physiologically [lo] , and biochemically [4, 15, 161, A net oxygen production was generally reported after several (2-4) hr of continuous illumination [ 171. In recent years, some groups have exposed etiolated leaves to intermittent light; a series of 1 msec flashes alternating with 15 min dark periods were used [6, 11, 10, 3, 21. In this case, it was found that 300 to 700 flashes (i.e. a total illumination of 0.3 to 0.7 set) induce the formation of chloroplasts containing “primary thylakoids”. These chloroplasts exhibit PMS catalyzed photophosphorylation, but no non-cyclic electron transport [lo] . Grana are not observed. Absence of some photosystem II properties was reported [3 ] . It was suggested [ 1 I] that short impulses of white light might induce the functioning of photosystem I only. In fact the development of PS II activity appears to require more than one brief illumination, even in the presence of excess chlorophyll in the leaf [7, 141. In this paper we report activation of photosystem II in flashed bean leaves when illuminated for the first time with continuous light, by measuring simultaneously oxygen production and fluorescence emission.

Light-stimulated synthesis of aminoacyl-tRNA synthetases in greening Euglena gracilis

Illumination of dark-grown Eugelena gracilis induces the formation of chloroplasts from nearly undifferentiated proplastids. This greening process is associated with remarkably increased activities of aminoacyl-tRNA synthetases which charge tRNAs from the blue-green alga Anacystis nidulans. After 48–72 h illumination a 10-fold enhancement of activity is observed for the enzymes binding leucine, lysine, valine, phenylalanine, methionine, tyrosine, arginine and aspartic acid to Anacystis tRNA. In these cases the light-induced rise of aminoacylation appears 50 % reduced if the enzymes are prepared from Euglena cells grown in the presence of 3 · 10 −3 M chloramphenicol or 2 · 10 −4 M nalidixic acid. In contrast, treatment of the cells for 3 days with 2 · 10 −5 M cycloheximide results in a stimulation of charging Anacystis tRNAs as compared with the enzymes from nontreated cells. Less stimulation by light and less inhibition by chloramphenicol or nalidixic acid is obtained for the enzymatic binding of alanine, glycine, serine, isoleucine, proline and threonine to Anacystis tRNAs. In some cases chromatography on hydroxylapatite columns can separate the crude enzyme preparations into two (or more) fractions with different acceptor specifities for the Anacystis tRNAs and the cytoplasmic tRNAs from Euglena cells. The results are discussed in relation to the site of synthesis of aminoacyl-tRNA synthetases in chloroplasts.

Nuclear dependency of chloroplast proteins in Acetabularia.

A membrane protein fraction, insoluble in water, has been prepared from Acetabularia chloroplasts. The greater part of these proteins could be dissolved in a phenol—formic acid—water mixture (2:1:1, v/v/v). The dissolved proteins were subjected to gel electrophoresis. The electrophoretic pattern was reproducible for one species.The protein patterns of A. calyculus and A. mediterranea were composed of 8 peaks common for both species and of at least 3 peaks which were species‐specific.By means of nuclear transplantation and implantation techniques it was shown that the species‐specific chloroplast‐proteins were nucleus‐dependent and therefore were coded by the nuclear DNA. Six weeks after the operation the patterns of the transplants and implants had changed into that of the nucleus donor species. It was concluded that the mRNA of the nucleus acceptor cell lost its stability under the influence of the implanted or transplanted nucleus.Both cycloheximide and chloramphenicol inhibited the incorporation of radioactive amino acids into the insoluble chloroplast‐protein fraction.The electrophoretic pattern of the labelled chloroplast‐proteins consisted of at least 3 peaks. Cycloheximide inhibited the incorporation into peak 3, whereas chloramphenicol affected the incorporation into the two other peaks. Isolated chloroplasts incorporated amino acids only into peaks 1 and 2. Peak 3 was absent in the pattern in vitro.The protein groups which were involved in the formation of chloroplasts were synthesized either by cytoplasmic 80 S or by 70 S chloroplast ribosomes. It was concluded that the autonomy of the chloroplasts was limited by the influence of the nucleus.

Chloroplast senescence in nongrowing tissue in darkness: Effects of temperature and age

Summary The senescence of chloroplasts in the leaf apical tissue was studied after transferring light-grown wheat plants to continuous darkness. In this system chloroplast senescence is uncoupled from chloroplast formation and can be followed as chlorophyll loss. This chloroplast senescence also occurs in absence of both cell division and expansion. The temperature-dependence for this chloroplast senescence was similar but less pronounced than the temperature dependence of senescence reported for invertebrate animals. The relative increase in rate per 10°C temperature rise was about 1·7 using different levels of chlorophyll loss as arbitrary, independent criteria of chloroplast senescence. In another series of experiments the temperature in darkness was always 20°C and the experimental variable was the age of the light-grown plants when transferred to darkness. The very youngest leaves showed an initial lag before chloroplast senescence set in, whereas the very oldest showed a slight acceleration of senescence within the first few days in continuous darkness. Over a wide range of middle-ages, however, the time-course of chloroplast senescence in darkness was the same. These results fail to support “Minot's paradox” as a generalization of an inverse relation between age and rate of senescence.

The effect of chloramphenicol and cycloheximide on lipid synthesis during chloroplast development in Euglena gracilis

The action of two inhibitors of protein synthesis, chloramphenicol and cycloheximide, on the production of lipids during chloroplast formation in the unicellular alga Euglena gracilis has been investigated. Under conditions where chloramphenicol prevented chlorophyll and chloroplast protein formation, the production of the galactolipids, monogalactosyl diacylglycerol, and digalactosyl diacylglycerol was also inhibited, although phospholipid synthesis was not affected. At low concentrations of cycloheximide (2.5 μg/5 × 10 6 cells), cell division was completely inhibited, but after an initial lag period of about 12 hr, chlorophyll production began at an accelerated rate. After 72 hr illumination, such cells contained substantially more chlorophyll than control cultures. Higher concentrations of cycloheximide increased the lag period before chlorophyll production began. Phospholipid production was severely inhibited in cycloheximide-treated cells, but the production of galactolipids showed the same pattern as that of chlorophyll, a lag phase followed by a period of accelerated synthesis. Cycloheximide also affected the molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol, and the fatty acid composition of the galactolipids. The galactolipids of cycloheximide-treated cells contained increased amounts of polyunsaturated fatty acids containing 16 or 18 carbon atoms.

Further similarities between chloroplast and bacterial ribosomes.

Protein synthesis by chloroplasts isolated under aseptic conditions from Phaseolus vulgaris leaves is inhibited by the bacterial antibiotics spectinomycin, lincomycin, and erythromycin; that by chloroplasts from Nicotiana tabacum leaves is inhibited by spectinomycin and lincomycin but not by erythromycin. Protein synthesis by cytoplasmic ribosomes from plants and animals is not inhibited by these compounds, nor is amino acid activation by the soluble fraction from bean chloroplasts. These results suggest that chloroplast ribosomes possess sites which bind several unrelated bacterial antibiotics and support the idea that chloroplasts originated from prokaryotic cells. These antibiotics may be useful in studying the process of chloroplast formation in intact cells.

Lipid metabolism of manganese-deficient algae. I. Effect of manganese deficiency on the greening and the lipid composition of Euglena gracilis Z.

The growth of photoautotrophic Euglena gracilis Z is strongly inhibited by manganese deficiency, whereas chlorophyll formation is not appreciably affected. The galactosyldiglyceride content of the manganese-deficient photo-autotrophic Euglena was about 40% lower on the basis of either chlorophyll content or dry weight. When dark-grown cultures of Euglena were grown photoheterotrophically in light sufficient for the greening of the cells, or photosynthesis, manganese deficiency resulted in a reduction of the cellular content of chlorophyll and galactosyldiglycerides to 40% of control values, indicating interference with chloroplast formation. The fatty acids of the photoheterotrophic manganese-deficient cells were mainly saturated, with an unusual accumulation (about 45%) of the total fatty acids) of myristic acid. In spite of this, the galactosyldiglycerides contain mainly unsaturated fatty acids. Ninety per cent of the fatty acids of the monogalactosyldiglyceride are unsaturated, including large amounts of alpha-linolenic acid. The ratio of chlorophyll to galactosyldiglyceride content of the cells was remarkably constant at all manganese deficiency levels.

雑 録 日本光生物学研究者名簿作成についてのお願い

雑 録 日本光生物学研究者名簿作成についてのお願い

Further similarities between chloroplast and bacterial ribosomes

Protein synthesis by chloroplasts isolated under aseptic conditions from Phaseolus vulgaris leaves is inhibited by the bacterial antibiotics spectinomycin, lincomycin, and erythromycin; that by chloroplasts from Nicotiana tabacum leaves is inhibited by spectinomycin and lincomycin but not by erythromycin. Protein synthesis by cytoplasmic ribosomes from plants and animals is not inhibited by these compounds, nor is amino acid activation by the soluble fraction from bean chloroplasts. These results suggest that chloroplast ribosomes possess sites which bind several unrelated bacterial antibiotics and support the idea that chloroplasts originated from prokaryotic cells. These antibiotics may be useful in studying the process of chloroplast formation in intact cells.

Nonphotosynthetic Retardation of Chloroplast Senescence by Light

Excised apical portions of green wheat leaf sections were treated with aminotriazole to prevent formation of new chloroplasts. Illumination retarded the decline in chlorophyll content per leaf section, the disintegration of chloroplast ultrastructure, and the loss of capacity for photosynthetic carbon fixation. We interpret these 3 effects of illumination as facets of a single light effect in retarding chloroplast senescence. This light effect in retarding chloroplast senescence has features differing from characteristics of photosynthetic carbon fixation. For example, A) application of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea did not decrease, and may have even slightly increased, the effectiveness of light; B) although the action spectrum contains peaks in the blue and red regions, it differs from the action spectrum for photosynthetic CO(2) assimilation in wheat; C) in nonphotosynthesizing tissue, application of sugars did not retard chloroplast senescence; D) light saturation was achieved by only a few hundred microwatts/cm(2). Considered together with the well-known light requirement for chloroplast formation, our results indicate that light has a dual, photomorphogenetic control in maintaining the green status of the plant by also exerting a second effect: retarding of senescence of chloroplasts already present.