Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.     

Complex origins of chloroplast membranes with photosynthetic machineries: multiple transfers of genes from divergent organisms at different times or a single endosymbiotic event?

The paradigm “cyanobacterial origin of chloroplasts” is currently viewed as an established fact. However, we may have to re-consider the origin of chloroplast membranes, because membranes are not replicated by their own. It is the genes for lipid biosynthetic enzymes that are inherited. In the current understandings, these enzymes became encoded by the nuclear genome as a result of endosymbiotic gene transfer from the endosymbiont. However, we previously showed that many enzymes involved in the synthesis of chloroplast peptidoglycan and glycolipids did not originate from cyanobacteria. Here I present results of comprehensive phylogenetic analysis of chloroplast enzymes involved in fatty acid and lipid biosynthesis, as well as additional chloroplast components related to photosynthesis and gene expression. Four types of phylogenetic relationship between chloroplast enzymes (encoded by the chloroplast and nuclear genomes) and cyanobacterial counterparts were found: type 1, chloroplast enzymes diverged from inside of cyanobacterial clade; type 2, chloroplast and cyanobacterial enzymes are sister groups; type 3, chloroplast enzymes originated from homologs of bacteria other than cyanobacteria; type 4, chloroplast enzymes diverged from eukaryotic homologs. Estimation of evolutionary distances suggested that the acquisition times of chloroplast enzymes were diverse, indicating that multiple gene transfers accounted for the chloroplast enzymes analyzed. Based on the results, I try to relax the tight logic of the endosymbiotic origin of chloroplasts involving a single endosymbiotic event by proposing alternative hypotheses. The hypothesis of host-directed chloroplast formation proposes that glycolipid synthesis ability had been acquired by the eukaryotic host before the acquisition of chloroplast ribosomes. Chloroplast membrane system could have been provided by the host, whereas cyanobacteria contributed to the genes for the genetic and photosynthesis systems, at various times, either before or after the formation of chloroplast membranes. The origin(s) of chloroplasts seems to be more complicated than the single event of primary endosymbiosis.

     

Preparation of Chloroplast DNA from Barley and Lettuce and Comparison of Restriction Fragments

Chloroplast DNA from three barley cultivars and from one lettuce cultivar was prepared from chloroplasts isolated by Conventional differential centrifugation. Barley chloroplast DNA size was sensibly lower (130 kpb) than lettuce chloroplast DNA (150 kpb). Chloroplast DNAs from the three barley cultivars showed similar restriction fragment patterns after digestion with: BamHI, EcoRI or HindIII. The lettuce chloroplast DNA restriction pattern was very different from the barley chloroplast DNA restriction pattern.     

植物叶绿体分裂的分子机制

叶绿体是植物细胞内一种重要的细胞器．它不仅是光合作用的场所,还是其它多种中间代谢的场所．叶绿体起源于蓝细菌,与其原核祖先类似,通过二分裂方式进行增殖．最近的研究表明,叶绿体的分裂装置包含原核起源和真核起源的蛋白质,它们在叶绿体的内膜内侧和外膜外侧协同作用以完成叶绿体的分裂．在过去十几年里,包括丝状温度敏感蛋白Z（FtsZ）、Min系统蛋白、质体分裂蛋白（PDV）和ARC蛋白等在内的多个叶绿体分裂相关组分被分离鉴定．本文简要介绍了叶绿体分裂装置各成员的发现、叶绿体被膜的收缩和叶绿体分裂位点的选择机制．另外,植物发育过程中叶绿体分裂可能受到细胞的控制,但目前对细胞如何调控叶绿体分裂知之甚少．本文对该领域的最新研究进展也进行了综述．     

Buoyant Density Studies of Chloroplast and Nuclear Deoxyribonucleic Acid from Control and 3-Amino-1,2,4-Triazole-treated Wheat Seedlings, Triticum vulgare

The isolation of chloroplast and nuclear DNA from dark- and light-grown, control- and 3-amino-1,2,4-triazole-treated wheat seedlings, Triticum vulgare, is described. Contrary to a previous report, we found that chloroplast and nuclear DNA had similar buoyant densities (1.702 grams per cubic centimeter) and that they could not be resolved by buoyant density centrifugation in CsCl. Difference in renaturation behavior of the chloroplast and nuclear DNA was used as the criterion for distinguishing one from the other. Only chloroplast DNA readily renatured whereas nuclear DNA renatured only slightly. Light-grown, 3-amino-1,2,4-triazole-treated plants were found to lack detectable quantities of chloroplast DNA whereas treated, dark-grown plants contained plastid DNA. We suggest that 3-amino-1,2,4-triazole affects the accumulation of chloroplast DNA by inhibiting the formation of chloroplast membranes, enzymes, and pigments.     

Effect of GDP on the interactions between chloroplast EF-Ts and chloroplast and E. coli EF-Tu

The effects of varying concentrations of GDP on the stability of homologous and heterologous EF-Tu:EF-Ts complexes formed with the elongation factors from the chloroplast of Euglena gracilis and from E. coli have been investigated. The complexes formed with chloroplast EF-Ts were significantly more stable to GDP-induced dissociation than those formed with E. coli EF-Ts. The complex between chloroplast EF-Tu and chloroplast EF-Ts required nearly 1,000-fold higher concentrations of GDP for dissociation than the complex between chloroplast EF-Tu and E. coli EF-Ts. The E. coli EF-Tu:chloroplast EF-Ts complex required nearly 100-fold higher levels of GDP for dissociation than the E. coli EF-Tu:E. coli EF-Ts complex.     

Phylogenetic congruence and discordance among one morphological and three molecular data sets from Pontederiaceae

A morphological data set and three sources of data from the chloroplast genome (two genes and a restriction site survey) were used to reconstruct the phylogenetic history of the pickerelweed family Pontederiaceae. The chloroplast data converged towards a single tree, presumably the true chloroplast phylogeny of the family. Unrooted trees estimated from each of the three chloroplast data sets were identical or extremely similar in shape to each other and mostly robustly supported. There was no evidence of significant heterogeneity among the data sets, and the few topological differences seen among unrooted trees from each chloroplast data set are probably artifacts of sampling error on short branches. Despite well-documented differences in rates of evolution for different characters in individual data sets, equally weighted parsimony permits accurate reconstructions of chloroplast relationships in Pontederiaceae. A separate morphology-based data set yielded trees that were very different from the chloroplast trees. Although there was substantial support from the morphological evidence for several major clades supported by chloroplast trees, most of the conflicting phylogenetic structure on the morphology trees was not robust. Nonetheless, several statistical tests of incongruence indicate significant heterogeneity between molecules and morphology. The source of this apparent incongruence appears to be a low ratio of phylogenetic signal to noise in the morphological data.     

盐胁迫下芦苇叶肉细胞超微结构的研究

对青藏高原柴达木盆地柯柯盐湖边盐碱地上生长的芦苇叶肉细胞的超微结构进行了研究,并以西宁地区非盐碱地上生长的芦苇作对照。结果表明：西宁地区的芦苇叶肉细胞的叶绿体呈椭圆形,其膜系统完整,基粒片层和基质片层发育良好。在盐碱地上生长的芦苇叶肉细胞的叶绿体呈圆形,叶绿体内出现较大的淀粉粒,并发现有线粒体嵌入叶绿体的现象。叶绿体的类囊体膨大,线粒体的嵴也有膨大的现象。在盐湖水中生长的芦苇叶肉细胞,叶绿体的类囊体排列紊乱、扭曲、松散。类囊体膜局部被破坏,部分类囊体膜解体,空泡化,甚至消失,一些溶解了的类囊体流进细胞质中。综上所述,芦苇叶肉细胞超微结构的变化是该植物适应柯柯盐湖地区盐渍、低温、低气压、强辐射等环境因子的结果。     

Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.     

Bt基因甜菜叶绿体转化载体构建及毒蛋白表达

利用植物叶绿体基因组在进化中高度保守的特点,根据烟草、菠菜、水稻叶绿体基因组全序列资料设计合成引物,PCR扩增并克隆了甜菜叶绿体两个重要功能基因rbcL和atpB(GenBank登录号分别为DQ067450和DQ067451),并以其作为定点整合外源基因的同源重组片段,构建了Bt基因CryIAc甜菜叶绿体定点转化载体pSKARBt,酶切鉴定表明:所构建载体符合预期设计。对克隆菌菌体总蛋白进行了生物杀虫试验,结果表明:Bt基因CryIAc能够在叶绿体特异性启动子及终止子的调控下表达,并对二龄末甘蓝夜蛾有很强的毒杀作用。该载体构建对培育甜菜高抗虫品种具有重要应用价值。叶绿体转化及后续工作正在进行中。     

ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

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.     

Water permeability of chloroplast envelope membranes. In vivo measurement by saturation-transfer NMR

In tulip tree (Liriodendron tulipifera) leaves, the proton NMR signal from chloroplast water is resolved from that of water in other leaf compartments. We used the saturation-transfer NMR method to measure the mean water molecule residence time within a chloroplast, (88 +/- 17) ms at 20 degrees C. From the measured chloroplast dimensions, we calculate an effective permeability coefficient of (9 +/- 2) X 10(-4) cm/s for the chloroplast envelope membrane. This is the first in vivo measurement of chloroplast water permeability.     

Euglena gracilis Chloroplast DNA Codes for Polyadenylated RNA

Polyadenylated RNA, isolated from total cellular RNA of photoautotrophically grown Euglena gracilis, comprised 2.1% of the total cellular RNA and contained 6.2% polyadenylic acid. Polyadenylated RNA, labeled in vitro with ¹²⁵I, hybridized at saturating levels to an average 7.7% of the chloroplast DNA. In the presence of excess chloroplast rRNA, hybridization of polyadenylated RNA was reduced, but was still observed at a level corresponding to 2.8% of the chloroplast DNA. Polyadenylic acid was not detected in mRNA prepared from chloroplast polyribosomes, indicating a level of less than 0.1% polyadenylic acid in mature chloroplast mRNA. Of the total RNA isolated from cytoplasmic polyribosomes, 2.0% contained polyadenylic acid. This latter polyadenylated RNA did not hybridize to chloroplast DNA.     

甘薯叶绿体表达载体构建及其融合基因在叶绿体中的瞬间表达

利用叶绿体基因组进化中高度保守的特点,根据烟草叶绿体基因组全序列设计引物,从甘薯(Ipomoea batatas)叶绿体基因组中克隆了2个相邻的功能基因rbcL(GenBank登录号为AY942199)和accD(GenBank登录号为AY942200),并以此作为定点整合外源基因的同源重组片段.以来自叶绿体基因组的强启动子Prrn和RpsbA-pro分别驱动选择标记基因aadA及phaC-gfp融合基因,构建成表达盒prrn-aadA-TpsbA-ter与RpsbA-pro-phaC-gfp-RpsbA-ter,然后将这2个表达盒串联在一起克隆进甘薯叶绿体同源片段中,获得甘薯叶绿体定点整合表达载体pSC-GFP.酶切分析证明,所构建的载体符合预期设计;采用该载体对甘薯叶片进行基因枪转化,结果显示,phaC-gfp融合基因可在叶绿体特异启动子和终止子的调控下在甘薯幼嫩叶片中瞬间表达,证明构建的载体pSC-GFP可用于甘薯叶绿体转化.     

A chloroplast genealogy of hordeum (poaceae): Long-term persisting haplotypes, incomplete lineage sorting, regional extinction, and the consequences for phylogenetic inference

To analyze reasons for inconclusive results of earlier chloroplast phylogenies in the grass genus Hordeum, we established a genealogy of chloroplast haplotypes by sequencing the trnL-trnF region in 875 individuals, covering all 31 species of the genus. Although the outcomes of phenetic and parsimony analyses of 88 haplotypes were ambiguous, a network approach showed that in Hordeum ancient chloroplast types co-occur with their descendants. Moreover, we found up to 18 different chloroplast haplotypes within a single species and up to 6 species sharing single haplotypes. Persisting polymorphisms together with incomplete lineage sorting occurred preferentially in the rapidly speciating New World taxa of the genus, where ancient chloroplast types have survived for at least 4 Myr. Lineages-through-time plots and a high number of missing chloroplast haplotypes indicated far-reaching extinction of chloroplast lineages in Europe and particularly the Mediterranean. Survival of these lineages in East Asia and North America resulted in chloroplast relationships that markedly differed from nuclear estimations of species relationships. Thus, even for the deepest splits in the genus, reaching back more than 9 Myr, no safe phylogenetic inference from chloroplast data is possible in Hordeum. The chloroplast genealogy, however, revealed biogeographic patterns and indicated processes involved in speciation in Hordeum. We conclude that the described phenomena are not restricted to Hordeum and that the knowledge of the chloroplast relationships within a genus is indispensable to prevent misinterpretation of phylogeographic data within single species.     

TRANSFER OF PROTEINS FROM THE CHLOROPLAST TO VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): A PATHWAY FOR DEGRADATION

Several chloroplast proteins were detected by immunoelectron microscopy within dense granules in cytoplasmic vacuoles in the alga Chlamydomonas reinhardtii Dangeard. Transfer from chloroplast to vacuoles of two major, pulse-labeled polypeptides, the large subunit of rubisco and the α subunit of ATPase, which are synthesized on chloroplast ribosomes, was demonstrated by the recovery of these polypeptides in vacuolar granules over a several-hour time period. The ultrastructure of cryofixed algal cells was examined to search for structures that would provide insight into the transfer of chloroplast proteins to vacuoles. Micrographs showed that the two membranes of the envelope were appressed, with no detectable intermembrane space, over most of the chloroplast surface. Protrusions of the outer membrane of the envelope were occasionally found that enclosed stroma, with particles similar in size to chloroplast ribosomes, but generally not thylakoid membranes. These observations suggest that chloroplast material, especially the stromal phase, was extruded from the chloroplast in membrane-bound structures, which then interacted with Golgi-derived vesicles for degradation of the contents by typical lysosomal activities. A protein normally targeted to vacuoles through the endomembrane system for incorporation into the cell wall was detected in Golgi structures and vacuolar granules but not the chloroplast.     

RIBOSOMES BOUND TO CHLOROPLAST MEMBRANES IN CHLAMYDOMONAS REINHARDTII

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein.