Adjustment of the tRNA population to the codon usage in chloroplasts.

In chloroplasts there is a correlation between the amounts of tRNAs specific for a given amino acid and the codons specifying this amino acid. Furthermore, for the amino acids coded for by more than one codon, the population of isoaccepting tRNAs is adjusted to the frequency of synonymous codons used in chloroplast protein genes. A comparison by two-dimensional gel electrophoresis of the tRNA populations extracted from chloroplasts and from chloroplast polysomes shows that all chloroplast tRNAs are involved in protein biosynthesis.     

Properties of chloroplasts isolated by phase partition

Summary Chloroplasts from spinach can be separated into at least three different populations by countercurrent distribution using polymer two-phase systems. The chloroplast particles of the three populations differ in protein/chlorophyll ratio, ultrastructure and metabolism. One population, peak I, consists of intact chloroplasts surrounded by the chloroplast envelope; the second population, peak II, consists of chloroplasts, which have lost their envelopes and much of their stromal material; the third population, peak III, consists of particles containing intact chloroplasts surrounded by a membrane-bound cytoplasmic layer including mitochondria and peroxisomes.Rapid batch procedures of peak I chloroplasts incorporated¹⁴C almost entirely into glycolate and intermediates of the Calvin cycle and starch synthesis. Only small amounts were found in sucrose and amino acids. On the other hand preparations of peak III chloroplasts gave a much broader spectrum of¹⁴C-labelled products. Sucrose, malate and some amino acids contained about 40% of the¹⁴C incorporated. It is concluded from these experiments that sucrose is formed not within the chloroplast but in the cytoplasm from intermediates exported by the chloroplast.The origin of peak III particles and their use for studying the cooperation between the chloroplast and the surrounding cytoplasm including mitochondria and peroxisomes is discussed.An invited article.     

Crystal structure of a conserved domain in the intermembrane space region of the plastid division protein ARC6

The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double‐membraned chloroplasts through binary fission. A key mediator of division‐complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division.     

茶树叶绿体及其蛋白的分离研究

茶树叶片叶绿体的有效分离纯化是进行茶树叶绿体代谢组学和蛋白质组学研究的基础.本文以茶树鲜叶为材料,通过叶绿体得率、希尔反应等纯度和完整度指标,比较了Percoll密度梯度离心法和蔗糖密度梯度离心法对叶绿体分离纯化的效果;通过蛋白质含量和SDS-PAGE电泳图谱,比较了涨破法和冻融法对叶绿体蛋白的提取效果.结果发现Per...     

Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone.

Nine different proteins were imported into isolated pea chloroplasts in vitro. For seven of these [the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), beta-subunit of ATP synthase, glutamine synthetase, the light-harvesting chlorophyll a/b binding protein, chloramphenicol acetyltransferase, and pre-beta-lactamase], a fraction was found to migrate as a stable high-molecular-weight complex during nondenaturing gel electrophoresis. This complex contained the mature forms of the imported proteins and the groEL-related chloroplast chaperonin 60 (previously known as Rubisco subunit binding protein). Thus, the stable association of imported proteins with this molecular chaperone is widespread and not necessarily restricted to Rubisco subunits or to chloroplast proteins. With two of the imported proteins (ferredoxin and superoxide dismutase), such complexes were not observed. It seems likely that, in addition to its proposed role in assembly of Rubisco, the chloroplast chaperonin 60 is involved in the assembly or folding of a wide range of proteins in chloroplasts.     

Chemical cross-linking and its effect on fatty acid synthetase activity in intact chloroplasts from Euglena gracilis

Intact chloroplasts were isolated from Euglena gracilis variety bacillaris, aliquots were exposed to several different chemical cross-linking reagents. The reagents penetrated the triple membrane of Euglena chloroplasts. This was shown by gradient acrylamide gel electrophoresis under denaturing conditions. The activity of the nonaggregated fatty acid synthetase of Euglena was located within the chloroplast stroma, and the effects of dimethylsuberimidate cross-linking on the activity of the enzyme system were examined. The acyl-carrier protein concentration in the chloroplast was measured at about 0.24 mM.     

The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var “Turkish Samsun” control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by “Samsun” chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.     

AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis

In plant cells, chloroplasts have essential roles in many biochemical reactions and physiological responses. Chloroplasts require numerous protein components, but only a fraction of these proteins are encoded by the chloroplast genome. Instead, most are encoded by the nuclear genome and imported into chloroplasts from the cytoplasm post-translationally. Membrane proteins located in the chloroplast outer envelope membrane (OEM) have a critical function in the import of proteins into the chloroplast. However, the biogenesis of chloroplast OEM proteins remains poorly understood. Here, we report that an Arabidopsis ankyrin repeat protein, AKR2A, plays an essential role in the biogenesis of the chloroplast OEM proteins. AKR2A binds to chloroplast OEM protein targeting signals, as well as to chloroplasts. It also displays chaperone activity towards chloroplast OEM proteins, and facilitates the targeting of OEP7 to chloroplasts in vitro. AKR2A RNAi in plants with an akr2b knockout background showed greatly reduced levels of chloroplast proteins, including OEM proteins, and chloroplast biogenesis was also defective. Thus, AKR2A functions as a cytosolic mediator for sorting and targeting of nascent chloroplast OEM proteins to the chloroplast.     

Biosynthetic cause of in vivo acquired thermotolerance of photosynthetic light reactions and metabolic responses of chloroplasts to heat stress

Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress.     

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein

Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.     

Co-association of cytochrome f catabolites and plastid-lipid-associated protein with chloroplast lipid particles

Distinguishable populations of lipid particles isolated from chloroplasts of yellow wax bean (Phaseolus vulgaris L. cv Kinghorn Wax) leaves have been found to contain plastid-lipid-associated protein (J. Pozueta-Romero, F. Rafia, G. Houlné, C. Cheniclet, J.P. Carde, M.-L. Schantz, R. Schantz [1997] Plant Physiol 115: 1185-1194). One population is comprised of plastoglobuli obtained from sonicated chloroplasts by flotation centrifugation. Higher density lipid-protein particles isolated from chloroplast stroma by ultrafiltration constitute a second population. Inasmuch as the stromal lipid-protein particles contain plastid-lipid-associated protein, but are distinguishable from plastoglobuli in terms of their lipid and protein composition, they appear to be plastoglobuli-like particles. Of particular interest is the finding that plastoglobuli and the higher density lipid-protein particles both contain catabolites of the thylakoid protein, cytochrome f. These observations support the view that there are distinguishable populations of plastoglobuli-like particles in chloroplasts. They further suggest that the formation of these particles may allow removal of protein catabolites from the thylakoid membrane that are destined for degradation as part of normal thylakoid turnover.     

Protein synthesis by chloroplasts during the senescence of barley leaves

Chloroplasts were isolated from senescent leaf segments of barley ( Hordeum vulgare L. var. Mozoncillo) and assayed for protein synthesis. Protein synthesis activity of the chloroplasts greatly increased after 10–20 h of incubation of leaf segments in the dark in spite of an intense degradation of chloroplast rRNA. The rise in the activity of protein synthesis was more pronounced when kinetin was present in the incubation medium. However, as deduced from SDS-polyacrylamide gel electrophoresis of the products, different proteins were synthesized under the two conditions of incubation of the leaf segments. The activity of protein synthesis of the chloroplasts decreased during the first hours of incubation of the leaf segments in the light.
Cutting and incubation in the dark of the leaf segments enhanced the synthesis of a few proteins also formed by chloroplasts in attached senescing leaves. Hormone and senescence treatments changed the type and the rate of the protein synthesized by chloroplasts, which suggests that hormones may control senescence through a modulation of the protein synthesized by the chloroplasts.     

Assembly of the Rieske iron-sulphur protein into the cytochrome bf complex in thylakoid membranes of isolated pea chloroplasts.

The assembly of the Rieske iron-sulphur protein into the cytochrome bf complex was examined following import of 35S-labeled precursor protein by isolated pea chloroplasts. Rieske protein assembled into the cytochrome bf complex was resolved from unassembled Rieske protein and from other membrane complexes by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. Four mutant forms of the Rieske protein were able to assemble into the cytochrome bf complex in isolated chloroplasts. These were a triple substitution mutant, C107S/H109R/C112S, replacing conserved residues involved in the ligation of the [2Fe-2S] centre; the mutant Delta45-52 which removed a glycine-rich region predicted to form a flexible hinge between the hydrophobic membrane-associated region and the hydrophilic lumenal domain; and mutants Delta168-173 and Delta177-179 which removed two C-terminal regions, which are highly conserved in chloroplast and cyanobacterial Rieske proteins. This indicates that the [2Fe-2S] cluster, the glycine-rich region and the C-terminal region are not essential for stable assembly of the Rieske protein into the cytochrome bf complex in isolated chloroplasts.     

Effect of Chloramphenicol on Formation of Chloroplast Structure and Protein During Greening of Etiolated Leaves of Phaseolus vulgaris

Greening of leaves of Phaseolus vulgaris in the presence of chloramphenicol inhibits formation of A) total chloroplast protein, B) an easily extractable fraction removed during isolation of chloroplasts in isotonic media by differential centrifugation, and C) the insoluble lamellar fraction which remains after extracting osmotically shocked freeze-dried plastids. The inhibition of insoluble chloroplast protein formation is correlated with decreased formation of lamellae and increased formation of vesicular structures. In contrast, chloramphenicol increases the formation of a fraction not removed during differential centrifugation, but removed by water extraction after osmotic shock and freeze-drying of chloroplasts. Analysis of this fraction by electrophoresis and column chromatography, indicates that the increased accumulation of this protein fraction is largely due to accumulation of a protein which is normally present in this fraction in small quantities. It was suggested that this protein may be a precursor which is normally incorporated into the lamellae. The protein extracted from freeze-dried chloroplasts of chloramphenicol treated chloroplasts contains a smaller proportion of one or more proteins than a similar extract of untreated plastids. However, per plastid, no such difference exists.     

快速提取、纯化叶绿体4.5SrRNA的方法

我们采用植物叶与热缓冲液、苯酚直接混合(约65℃)匀浆,离心抽提和乙醇沉淀后,得到植物叶总RNA。经聚丙烯酰胺凝胶电泳分离、纯化,即可得到叶绿体4.5S rRNA,此法不仅操作简单,而且得率高。 同时,经过对同一植物的不同组织或不同细胞组分,如根、细胞质、叶绿体和叶绿体核糖体小分子RNA的提取与鉴定,以简便的方法证明了4.5S rRNA是叶绿体核糖体成份,也证明了我们所采用的提取、纯化4.5SrRNA方法的可靠性。     

Purification, subunit structure and immunological comparison of fructose-bisphosphate aldolases from spinach and corn leaves

The cytosol and chloroplast fructose-bisphosphate aldolases from spinach leaves were separated by ion-exchange chromatography on DEAE-cellulose, and were purified by subsequent affinity chromatography on phosphocellulose to apparent homogeneity as judged from polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two aldolases had specific activities of 7.2 and 7.8 units mg protein-1. Molecular weight determinations by electrophoresis in sodium dodecyl sulfate gels and by sedimentation velocity centrifugation in sucrose gradients showed that the aldolases contained four subunits of Mr 38 000 and 35 000, respectively. Antibodies against the cytosol and chloroplast aldolase from spinach leaves were raised in a guinea pig and in a rabbit, respectively. In the Ouchterlony double-diffusion test, the two aldolases did not cross-react. A small degree of cross-reaction was observed by a test in which immune complexes were adsorbed to a solid-phase support (Staphylococcus aureus Cowan I cells) and nonbound enzyme activity was determined after centrifugation. These results imply major structural differences between the two spinach leaf aldolases. Only one major aldolase could be resolved on DEAE-cellulose from corn leaves. The aldolase was purified and had a specific activity of 6.4 units X mg protein-1. The corn leaf aldolase cross-reacted with the antiserum raised against the chloroplast enzyme from spinach leaves, but not with the other antiserum. Thus, the corn leaf aldolase could be identified as a chloroplast enzyme. Since aldolase activity is mostly restricted to the bundle sheath cells of corn leaf, it was concluded that it is compartmentalized in the chloroplasts of these cells but not in chloroplasts of the mesophyll cells.     

Effect of Manganese Deficiency on Chloroplasts of Lemon Leaves

Manganese deficiency in chloroplasts of Eureka lemon leaves resulted in 23% and 40% increase of chloroplast nitrogen and protein, respectively, on a chlorophyll unit basis. Acrylamide gel electrophoresis carried out on extracts of these chloroplasts disclosed also qualitative differences between the normal and deficient leaves. Calculated on chloroplast N basis there is an increase of 17% in the chloroplast protein under Mn deficiency. This increase apparently indicates a more intense protein synthesis in the Mn deficient chloroplasts. Hill activity of the –Mn leaves was about one-third of the analog control leaves. Manganese infiltration into detached but intact leaves restored the activity in the –Mn leaves up to 70% of the control. Lemon leaves affected by other macro- and micro- nutrient deficiencies did not respond to the manganese infiltration; therefore, the use of this infiltration method is suggested for the evaluation of the manganese nutrition status of citrus and probably other higher plants.     

Chloroplast envelope proteins are encoded by the chloroplast genome of Chlamydomonas reinhardtii.

To characterize envelope proteins encoded by the chloroplast genome, envelopes were isolated from Chlamydomonas reinhardtii cells labeled with [35S] sulfate while blocking synthesis by cytoplasmic ribosomes. One and two-dimensional gel electrophoresis of envelopes and fluorography revealed four highly labeled proteins. Two with masses of 29 and 30 kDa and pI 5.5 were absent from the stroma and thylakoid fractions, while the others at 54 kDa, pI 5.2 and 61 kDa, pI 5.4 were detected there in smaller amounts. The 29- and 30-kDa proteins were associated with outer envelope membranes separated from inner envelope membranes after chloroplast lysis in hypertonic solution. A 32-kDa protein not labeled by [35S]sulfate was found exclusively in the inner membrane fraction, suggesting the existence of a phosphate translocator in C. reinhardtii. To identify envelope proteins exposed on the chloroplast surface, isolated active chloroplasts were surface-labeled with 125I and lactoperoxidase. The 54-kDa, pI 5.2 protein as well as a protein corresponding to either of the 29- or 30-kDa proteins described above were among the labeled components. These results show that envelope proteins of C. reinhardtii are encoded by the chloroplast genome and two are located on the outer envelope membranes.     

Protein import into cyanelles and complex chloroplasts

Higher-plant, green and red algal chloroplasts are surrounded by a double membrane envelope. The glaucocystophyte plastid (cyanelle) has retained a prokaryotic cell wall between the two envelope membranes. The complex chloroplasts of Euglena and dinoflagellates are surrounded by three membranes while the complex chloroplasts of chlorarachniophytes, cryptomonads, brown algae, diatoms and other chromophytes, are surrounded by 4 membranes. The peptidoglycan layer of the cyanelle envelope and the additional membranes of complex chloroplasts provide barriers to chloroplast protein import not present in the simpler double membrane chloroplast envelope. Analysis of presequence structure and in vitro import experiments indicate that proteins are imported directly from the cytoplasm across the two envelope membranes and peptidoglycan layer into cyanelles. Protein import into complex chloroplasts is however fundamentally different. Analysis of presequence structure and in vitro import into microsomal membranes has shown that translocation into the ER is the first step for protein import into complex chloroplasts enclosed by three or four membranes. In vivo pulse chase experiments and immunoelectronmicroscopy have shown that in Euglena, proteins are transported from the ER to the Golgi apparatus prior to import across the three chloroplast membranes. Ultrastructural studies and the presence of ribosomes on the outermost of the four envelope membranes suggests protein import into 4 membrane-bounded complex chloroplasts is directly from the ER like outermost membrane into the chloroplast. The fundamental difference in import mechanisms, post-translational direct chloroplast import or co-translational translocation into the ER prior to chloroplast import, appears to reflect the evolutionary origin of the different chloroplast types. Chloroplasts with a two-membrane envelope are thought to have evolved through the primary endosymbiotic association between a eukaryotic host and a photosynthetic prokaryote while complex chloroplasts are believed to have evolved through a secondary endosymbiotic association between a heterotrophic or possibly phototrophic eukaryotic host and a photosynthetic eukaryote.     

The 68 kDa DNA compacting nucleoid protein from soybean chloroplasts inhibits DNA synthesis in vitro

Nucleoids were purified from chloroplasts of dividing soybean cells and their polypeptide composition analyzed by SDS-polyacrylamide gel electrophoresis. Of the 15–20 nucleoid-associated polypeptides, several demonstrated DNA binding activity. Upon disruption of the nucleoids with high concentrations of NaCl, a subset of these proteins and the majority of chloroplast DNA were recovered in the supernatant after centrifugation. Removal of the salt by dialysis resulted in formation of nucleoprotein complexes resembling genuine nucleoids. Purification of these structures revealed three major proteins of 68, 35 and 18 kDa. After purification of the 68 kDa protein to homogeneity, this protein was able to compact purified chloroplast DNA into a nucleoid-like structure in a protein concentration-dependent fashion. Addition of the 68 kDa protein to an in vitro chloroplast DNA replication system resulted in complete inhibition of nucleotide incorporation at concentrations above 300 ng of 68 kDa protein per g of template DNA. These results led to in situ immunofluorescence studies of chloroplasts replicating DNA which suggested that newly synthesized DNA is not co-localized with nucleoids. Presumably, either the plastid replication machinery has means of removing nucleoid proteins prior to replication or the concentration of nucleoid proteins is tightly regulated and the proteins turned over in order to allow replication to proceed.