几种因子对离体豌豆叶绿体蛋白质合成的影响

利用制备的豌豆完整叶绿体研究了离体条件下蛋白质合成的条件。结果表明：叶绿体蛋白质合成的饱和光强为450μmol-2s－1,合成的速率在最初5min内最大,此后随时间延长而合成速率下降;K 对蛋白质合成有促进作用,其最适浓度为30-40mmol/L,进一步增加浓度其促进作用反而降低;Mg2 在1mmol／L以下对蛋白质合成有轻微的促进作用,当浓度超过1．5mmol/L则开始产生明显的抑制;叶绿体的蛋白质合成随着外源氨基酸浓度的增加而很快地增加,但赵过200μmol/L以后蛋白质合成随浓度增加而有所降低。DCMU抑制叶绿体蛋白质的合成,当浓度达10μmol/L时,其抑制作用达41％。荧光自显影结果表明,叶绿体合成的主要问质蛋白为Rubisco大亚基,合成的类囊体膜蛋白中以32kD蛋白较为明显。     

In vitro Protein Synthesis by Plastids of Phaseolus vulgaris. III. Formation of Lamellar and Soluble Chloroplast Protein

Chloroplasts from leaves of plants which had been grown in the dark, and then illuminated for 12 hours were isolated, and allowed to incorporate ¹⁴C-leucine into protein, and the products of this incorporation were studied. Lamellar and soluble proteins are the principal products, and are formed in about equal amounts. Only some of the soluble proteins become heavily labeled. Those with highest specific activity have a molecular weight of the order of 140,000, while the higher molecular weight Fraction I protein has a much lower specific activity. The soluble protein as a whole does not serve as a precursor for the lamellar protein, and vice-versa, although a precursor-product relationship between a minor component of the soluble fraction and the lamellar fraction has not been ruled out. The relative protein synthesizing capabilities of chloroplasts and mitochondria are discussed with reference to the data presented.     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings V. Protein Synthesis in Vivo

Incorporation of ¹⁴C-amino acids into proteins in radish cotyledonswas suppressed by 4-thiouridine (4SU) culture. The inhibitoryeffect of 4SU was similar to that of chloramphenicol. 4SU culturedid not reduce the content of ferredoxin (Fd) and the labelinginto Fd significantly, but it did decrease the content and thesynthesis of ribulose bisphosphate carboxylase (RuBPCase). Thesynthesis of thylakoid chlorophyll-proteins I and II also wasinhibited by 4SU culture. In 4SU-cultured seedlings, the ratioof labeling into the large and small subunits of RuBPCase andthat into the two chlorophyll-proteins were the same as thosein the controls grown without 4SU. (Received September 29, 1980; Accepted January 27, 1981)     

小麦叶片中叶绿体细胞分裂素结合蛋白的定位

小麦叶片中叶绿体细胞分裂素结合蛋白的定位张华敏,刘愚,王美琪,沈允钢（中国科学院上海植物生理研究所,上海２０００３２）关键词：小麦叶片细胞,ＣＴＫ结合蛋白,放射自显影,胶体金自从Ｂｅｒｒｉｄｇｅ等（１９７０）首次在高等植物的核糖体上发现了细胞分裂素（...     

The Formation of Ribulose Diphosphate Carboxylase Protein during Chloroplast Development in Barley

Ribulose 1,5-diphosphate carboxylase is synthesized in barley leaves growing in the dark. Upon illumination there is a marked increase in the rate of synthesis of the enzyme. The specific activity of the enzyme expressed as cpm incorporated into phosphoglyceric acid per μg of fraction I protein, after isolation shows no change either during dark growth or greening. During early stages of illumination of 7 day dark grown leaves with 320 foot-candles the enzymic activity in the water soluble protein fraction of the leaf shows a short term decline after 15 min which lasts for 30 min. Leaves greening at 2 foot-candles show a similar decline which is shifted to a time between the fourth and eighth hr after the onset of illumination.     

Chloroplast Division and DNA Synthesis in Light-grown Wheat Leaves

Light-grown 7-day-old wheat seedlings (Triticum aestivum, var. Maris Dove) showed an increase of 200% in plastids per cell between 1.7 and 4.5 centimeters from the leaf base. This increase was the result of divisions of young chloroplasts at various stages of development, and was well separated in distance, and therefore in time from the region of cell division in the basal meristem. [³H]Thymidine was incorporated into plastid DNA throughout the zone of plastid division, but not above it.     

Synthesis of an Atrazine-Binding Protein of the PSII Reaction Center in Isolated Wheat Etiochloroplasts

Isolated wheat (Triticum aestivum L. cv Norin 61) etiochloroplastssynthesized membrane polypeptides of 34, 33 and 30 kDa whichwere resolved by lithium dodecyl sulfate polyacrylamide gelelectrophoresis at 4?C. One-dimensional peptide-mapping analysis,as well as differential labelling with (³H)-lysine or (³⁵S)-methionine,showed that the 34- and 30-kDa polypeptides are atrazine-bindingproteins of the PSII reaction center. ¹Present address: Research Center for Molecular Genetics, HokkaidoUniversity, Sapporo 060, Japan. (Received March 9, 1987; Accepted June 29, 1987)     

Chloroplast Dedifferentiation in Mechanically Isolated Asparagus Cells during Culture Initiation

Mechanically isolated asparagus (Asparagus officinalis) mesophyll cells dedifferentiate and divide when cultured in the dark in a medium containing sucrose. A strong correlation was observed between the onset of cell division and a loss of photosynthetic capacity. For the first 8 to 9 d of culture, there was no change in chloroplast size or morphology. However, following this period, the chloroplasts divided to form smaller proplastid-like structures. The gross chlorophyll content of the cell population did not change, suggesting that the loss of photosynthetic potential was not by senescence. Northern analysis showed that mRNA of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase was undetectable within 1 d postisolation, which was quicker than in dark-treated plants. The mRNA of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase decreased to low levels within 2 d of cell isolation. Both the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase protein showed a gradual reduction in abundance, falling to basal levels by days 6 to 7, which coincided with the onset of rapid cell division. A similar trend was observed with chloroplast rRNA molecules, which decreased to basal levels by day 6 in culture.     

Synthesis of Chloroplast Proteins during Germination and Early Development of Cucumber

Cell-free protein synthesizing systems have been used to study the developmental changes in the synthesis of chloroplast proteins in the cotyledons of cucumber seedlings grown in the light or in the dark. Escherichia coli and wheat germ in vitro protein synthesizing systems have been used to assay the changes in the levels of the mRNA's coding for ribulose 1,5-bisphosphate carboxylase (RuBPCase). The large subunit of cucumber RuBPCase has been identified among the translation products of the E. coli system. The wheat germ system translates the cucumber mRNA coding for the small subunit of RuBPCase to produce a 25,000 molecular weight precursor polypeptide. Plastids isolated from light-grown cotyledons were used to study developmental changes in their capacity to synthesize protein. The data obtained indicate that in the light there is an initial 48-hour period of accumulation of the mRNA's coding for the large and small subunits of RuBPCase, coupled with an increase in the capacity of the isolated plastids to synthesize protein. This is followed by a decline. This decline is not reflected in the accumulation of RuBPCase in the cotyledons which remains constant over the period of study.     

Protein Targeting into Secondary Plastids1

ABSTRACT. Most of the coding capacity of primary plastids is reserved for expressing some central components of the photosynthesis machinery and the translation apparatus. Thus, for the bulk of biochemical and cell biological reactions performed within the primary plastids, many nucleus‐encoded components have to be transported posttranslationally into the organelle. The same is true for plastids surrounded by more than two membranes, where additional cellular compartments have to be supplied with nucleus‐encoded proteins, leading to a corresponding increase in complexity of topogenic signals, transport and sorting machineries. In this review, we summarize recent progress in elucidating protein transport across up to five plastid membranes in plastids evolved in secondary endosymbiosis. Current data indicate that the mechanisms for protein transport across multiple membranes have evolved by altering pre‐existing ones to new requirements in secondary plastids.     

Behavior of Chloroplast Nucleus during Chloroplast Development and Degeneration in Chlamydomonas reinhardii

Changes in morphology of chloroplast nuclei (cp-nuclei), totalcp-DNA content, number of cp-nuclei, oxygen-evolution activityand chlorophyll (a and b) content were examined during the degenerationand development of chloroplasts, using Chlamydomonas reinhardiicells which had been incubated on solid medium for various periods. Under 4'-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy,each cell that had been incubated for 7 days had one cell nucleus,one cup-shaped chloroplast and about 10 small, dispersed cp-nucleiin the chloroplast. One day after incubation of these cellson fresh medium, the cell volume and cp-nuclei increased insize 2-3 fold, but rapidly decreased in size after cell division.After about 7 days of incubation, cells ceased to divide andcp-nuclei began to associate with each other. At about 20 daysthey formed a ring-shaped structure surrounding the pyrenoid,followed by condensation into one cp-nuclear particle near thepyrenoid. When 41-day-old cells, having only one cp-nucleus,were reinoculated on fresh solid medium, the cp-nucleus increasedin size 2–3 fold, divided into several cp-nuclear particlesand then dispersed into the chloroplast, forming a bead-likestructure, before cell division. From microscopic fluorometry,a 4-fold increase in total cp-DNA content per chloroplast, withoutan increase in the number of cp-nuclear particles per chloroplast,occurred one day after the start of the experiment and one dayafter reinoculation of 41-day-old cells onto fresh medium. Theprocess of condensation of dispersed cp-nuclear particles intoone cp-nucleus during degeneration of the chloroplast was notaccompanied by any change in total cp-DNA content per chloroplast.A large peak of oxygen-evolution (0.6–0.9 pmoles/cell/hour)was seen one day after inoculation and reinoculation of thecells. The chlorophyll content (a+b) was high (1.2–2.2pg/cell) during the first week of incubation, after which itgradually decreased. (Received December 18, 1985; Accepted April 2, 1986)     

Development of Chloroplast in Dark-Grown Wheat Seedlings Irradiated at Elevated Temperature

Elevated temperature inhibited the accumulation of chlorophyll and photosynthetic proteins, and the development of photochemical activity, however, carotenoids continued to accumulate. Signal transduction pathway involved in protochlorophyllide oxidoreductase was unaffected by elevated temperature of 38°C. Two-dimensional gel electrophoresis of stroma proteins showed similar patterns in the dark-grown seedlings and seedlings irradiated at elevated temperature, although some low molecular mass proteins accumulated at 38°C. In contrast, seedlings irradiated at 25°C showed complex pattern of proteins. Hence the development of chloroplast and its associated functions during irradiation of etiolated seedlings are inhibited by elevated temperature.     

The Circadian Oscillator Coordinates the Synthesis of Apoproteins and Their Pigments during Chloroplast Development

Greening has been studied at circadian times of maximal and minimal levels of mRNA for the light-harvesting chlorophyll a/b binding protein in photosystem II (Cab mRNA) after circadian synchronization of etiolated barley plantlets (Hordeum vulgare cv Apex) by heat-shock treatments. It was found that greening occurs faster and without a lag period when illumination was started at the time of maximal Cab mRNA accumulation. This holds true for the rate of accumulation of Cab and early light-inducible protein mRNAs, the levels of their correspondent proteins, and the levels of chlorophyll a and b. When illumination was started at the time of Cab mRNA minimum, a lag in the appearance of all components mentioned above was observed. Under these conditions, the lag in chlorophyll b accumulation was by far more pronounced than that found for chlorophyll a. The circadian oscillation in the capacity of chlorophyll synthesis appears to be controlled via [delta]-aminolevulinic acid ([delta]-ALA) synthesis. [delta]-ALA accumulation after levulinic acid treatment is itself under circadian control; the maxima in stationary concentrations coincide with those of Cab mRNA levels. The amounts of protochlorophyllide and photoconvertible protochlorophyllide showed only minor differences between circadian minima and maxima, the levels being slightly lower during the time of minimum.     

Induced Changes in Chloroplast Protein Accumulation during Heat Bleaching in Euglena gracilis

When growing cultures of light-grown Euglena gracilis Z are exposed to slightly elevated temperatures (33°C) there is a time-dependent decrease in chlorophyll (bleaching) and a gradual transformation of chloroplasts into rudimentary plastids. A study was undertaken whose primary objective was to document major changes in polypeptide composition in the stroma and in thylakoids of cells that have been exposed to the bleaching temperature for up to 57 hours. A novel polypeptide of about 60,000 to 63,000 M_r whose function is presently unknown, accumulates in the stroma and in thylakoids in response to growth at the bleaching temperature. The levels of the large and small subunit of ribuolosebisphosphate carboxylase, on the other hand, decrease to very low levels at about 33 hours and remain very low for the duration of the temperature treatment. Of two polypeptides associated with the light-harvesting chlorophyll-protein complex of photosystem II (28,000 and 24,500 M_r) only the level of the smaller polypeptide decreases at the elevated temperature. The levels of 28,000 M_r species remain virtually unchanged throughout the temperature treatment period. Changes in chloroplast polypeptide composition were also studied in cells that were allowed to recover at room temperature from an initial treatment at 33°C. Bleaching Euglena could provide a useful tool for studying the interaction between the nucleus and chloroplast genetic system that govern the development and maintenance of this vital organelle to plants.     

蛋白质向叶绿体的转运

对近年来叶绿体蛋白质前导肽序列、叶绿体被膜中的蛋白质转运器、监护蛋白在蛋白转运过程中的作用、蛋白质导入叶绿体的途径、前体蛋白的加工的研究进展进行了介绍和评述     

Differential Synthesis of Photosystem Cores and Light-Harvesting Antenna during Proplastid to Chloroplast Development in Spirodela oligorrhiza

Proplastids and etioplasts are common starting points for monitoring chloroplast development in higher plants. Although proplastids are the primary precursor of chloroplasts, most proplastid to chloroplast systems are cumbersome to study temporally. Conversely, the etioplast to chloroplast transition is initiated by light and is readily examined as a function of time. Etioplasts, however, are found mostly in plants germinated in the dark and are not an obligatory step in chloroplast development. We have chosen to study chloroplast ontogeny in Spirodela oligorrhiza (Kurtz) Hegelm (a C₃-monocot) because of its unique ability to grow indefinitely in the dark. Ultrastructural, physiological, and molecular evidence is presented in support of a temporal, light-triggered proplastid to chloroplast transition in Spirodela. The dark-grown plants are devoid of chlorophyll, and upon illumination synchronously green over a 3- to 5-day period. Synthesis of chloroplast proteins involved in photosynthesis is coincident with thylakoid assembly, chlorophyll accumulation, and appearance of CO₂ fixation activity. Interestingly, the developmental sequence in Spirodela was slow enough to reveal that biosynthesis of the D1 photosystem II reaction center protein precedes biosynthesis of the major light-harvesting antenna proteins. This, coupled with the high chlorophyll a/b ratio observed early in development, indicated that reaction center assembly occurred prior to accumulation of the light-harvesting complexes. Thus, with Spirodela one can study proplastid to chloroplast conversions temporally in higher plants and follow the process on a time scale that enables a detailed dissection of plastid maturation processes.     

Plastidic Isoprenoid Synthesis during Chloroplast Development : Change from Metabolic Autonomy to a Division-of-Labor Stage

The chloroplast isoprenoid synthesis of very young leaves is supplied by the plastidic CO₂ → pyruvate → acetyl-coenzyme A (C₃ → C₂) metabolism (D Schulze-Siebert, G Schultz [1987] Plant Physiol 84: 1233-1237) and occurs via the plastidic mevalonate pathway. The plastidic C₃ → C₂ metabolism and/or plastidic mevalonate pathway of barley (Hordeum vulgare L.) seedlings changes from maximal activity at the leaf base (containing developing chloroplasts with incomplete thylakoid stacking but a considerable rate of photosynthetic CO₂-fixation) almost to ineffectivity at the leaf tip (containing mature chloroplasts with maximal photosynthetic activity). The ability to import isopentenyl diphosphate from the extraplastidic space gradually increases to substitute for the loss of endogenous intermediate supply for chloroplast isoprenoid synthesis (change from autonomic to division-of-labor stage). Fatty acid synthesis from NaH¹⁴CO₃ decreases in the same manner as shown for leaf sections and chloroplasts isolated from these. Evidence has been obtained for a drastic decrease of pyruvate decarboxylase-dehydrogenase activity during chloroplast development compared with other anabolic chloroplast pathways (synthesis of aromatic amino acid and branched chain amino acids). The noncompetition of pyruvate and acetate in isotopic dilution studies indicates that both a pyruvate-derived and an acetate-derived compound are simultaneously needed to form introductory intermediates of the mevalonate pathway, presumably acetoacetyl-coenzyme A.     

Chloroplast Protein Synthesis in the Chromophytic Alga Olisthodiscus luteus: Cell Cycle Analysis

This study represents the first report on chloroplast protein synthesis during the synchronous cell growth of a chromophytic (chlorophyll a,c) plant. When the unicellular alga Olisthodiscus luteus is maintained on a 12-hour light:12-hour dark cycle, cell and chloroplast number double every 24 hours. A temporal separation between these two events occurs. Measurements of chloroplast and total cellular protein values suggest that polypeptide synthesis occurs mainly in the light portion of the cell cycle, and pulse chase studies demonstrate that chloroplast proteins made in the light are not degraded in the dark. Data support the following conclusions: (a) a similar complement of chloroplast DNA coded proteins is made at all phases of the light portion of the cell cycle, and (b) chloroplast protein synthesis is a light rather than a cell cycle mediated response.