Isolation Of Chloroplasts Research Articles

An improved method for chloroplast genome sequencing in non-model forest tree species

Chloroplast genomes can provide a large amount of information and resources for use in studies on plant evolution and molecular ecology. However, a rapid and efficient method for obtaining chloroplast genome sequences is still lacking. In this study, we report a modified method for the isolation of intact chloroplasts, which needs less than 0.5 g leaf material. Coupled with rolling circle amplification (RCA), next-generation sequencing, and a pipeline combining de novo assembly and reference-guided assembly (RGA), we successfully obtained a complete chloroplast genome for the non-model forest tree species, evergreen oak Quercus spinosa, with as many as 36 % of the sequence reads mapped to the chloroplast genome. The Q. spinosa cpDNA is 160,825 bp in length and codes for 134 genes (89 protein coding, 8 ribosomal RNAs (rRNAs), and 36 distinct transfer RNAs (tRNAs)). The genome organization and arrangement are similar to those found among most angiosperm chloroplast genomes. Our inexpensive and efficient protocol can be applied to the reconstruction of chloroplast genomes for plant evolutionary studies, especially in non-model tree species.

Isolation of Chloroplasts from Plant Protoplasts.

Chloroplasts can be isolated from higher plants directly following homogenization; however, the resulting yield, purity, and intactness are often low, necessitating a large amount of starting material. This protocol is optimized to produce a high yield of pure chloroplasts from isolated Arabidopsis protoplasts. The two-part method is a simple, scaled-down, and low-cost procedure that readily provides healthy mesophyll protoplasts, which are then ruptured to release intact chloroplasts. Chloroplasts isolated using this method are competent for use in biochemical, cellular, and molecular analyses.

Development of polymorphic chloroplast DNA markers for the endangered tree Eusideroxylon zwageri through chloroplast isolation and next-generation sequencing

Borneo ironwood (Eusideroxylon zwageri) is a tall tropical tree that is threatened by illegal logging and exploitation due to its economic value. In the present study, approximately 10,618 bp of E. zwageri chloroplast DNA were sequenced genome-wide and 16 polymorphic markers of chloroplast DNA were developed, using next-generation sequencing technology. In total, 26 nucleotide substitutions, 3 indels, 1 sequence inversion, and 1 mononucleotide repeat variation were detected in samples of 10 trees collected from 5 populations in Kalimantan, Indonesia. These markers are a powerful tool that may be used to describe the phylogeographical genetic structure of E. zwageri and will be essential for the conservation and management of this endangered tree.

Next generation sequencing of total DNA from sugarcane provides no evidence for chloroplast heteroplasmy

BackgroundThe chloroplast genome of plants has been frequently sequenced using chloroplast DNA derived by techniques involving chloroplast isolation and or by PCR amplification using primer sequences targeted to amplify the chloroplast genome. Using these approaches, chloroplast heteroplasmy, described as variations in the chloroplast sequence within an individual plant, has been reported in many plant species. More recently, next generation sequencing (NGS) technologies have allowed chloroplast genome sequences to be extracted from shotgun sequences of total plant DNA. ResultsHere, we used DNA preparations varying in nuclear, mitochondrial and chloroplast enrichment to explore the potential to distinguish genuine chloroplast heteroplasmy from apparent heteroplasmy due to sequence-variant homologues of chloroplast genome sequences inserted in nuclear or mitochondrial genomes. Application of NGS to the whole sugarcane genome followed by read mapping analysis of the complex sugarcane system allowed the assembly of a complete chloroplast genome sequence of sugarcane cv. Q155. Variant analysis showed that they were present only at frequencies that could be attributed to homologues of chloroplast sequences inserted in the nucleus or mitochondria. ConclusionsThe result suggests that earlier reports of heteroplasmy in chloroplasts may have been due to contaminating sequences from other genomes (nuclear or mitochondrial) in chloroplast preparations or specific amplification of sequences from these genomes. This demonstrates that the ability to evaluate sequence abundance avoids the risks of attributing a chloroplast gene homologue from the nucleus or mitochondria to the chloroplast.

Chloroplast isolation and affinity chromatography for enrichment of low-abundant proteins in complex proteomes.

Detailed knowledge of the proteome is crucial to advance the biological sciences. Low-abundant proteins are of particular interest to many biologists as they include, for example those proteins involved in signal transduction. Recent technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS). However, the dynamic range in protein abundance still forms a fundamental problem that limits the detection of low-abundant proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of other proteins at an abundance several-fold higher in order of magnitude. Therefore, specific enrichment strategies are required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundant proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.

Phosphorus compounds, proteins, nuclease and acid phosphatase activities in isolated spinach chloroplasts

This paper deals with attempts to elaborate a simple method of spinach chloroplast isolation ensuring a high proportion of intact chloroplasts. We obtained 3 preparations of isolated chloroplasts. Several preliminary analyses of the obtained chloroplast fraction were also performed. Phosphorus compounds, total protein and the enzyme activities of RNase, DNase and GPase were determined. We found: 0,36-0,59% of RNA, 0,19-0,24% of DNA, 2,1-2,9% of phospholipids and 26-28% of protein. RNase activity was very high.

Cosuppression of the chloroplast localized molecular chaperone HSP90.5 impairs plant development and chloroplast biogenesis in Arabidopsis.

BackgroundHSP90.5 is a chloroplast localized HSP90 family molecular chaperone in Arabidopsis, and it has been implicated in plant abiotic stress resistance, photomorphogenesis and nuclear-encoded protein import into the chloroplast. However, how these processes are controlled by HSP90 is not well understood. To understand the role of HSP90.5 in chloroplast function and biogenesis, in this study, we generated transgenic Arabidopsis plants that overexpress a C-terminally FLAG-tagged HSP90.5. By characterizing three HSP90.5 cosuppression lines, we demonstrated the essential role of HSP90.5 in plant growth and chloroplast biogenesis.ResultsImmunoblotting and quantitative PCR analyses revealed three independent HSP90.5 cosuppressing transgenic lines. All three cosuppression lines displayed a certain degree of variegated phenotype in photosynthetic tissues, and the cosuppression did not affect the expression of cytosolic HSP90 isoforms. HSP90.5 cosuppression was shown to be developmentally regulated and occurred mostly at late developmental stage in adult leaves and inflorescence tissues. HSP90.5 cosuppression also caused significantly reduced rosette leaf growth, transient starch storage, but did not affect rosette leaf initiation or inflorescence production, although the fertility was reduced. Isolation of chloroplasts and size exclusion chromatography analysis indicated that the FLAG at the HSP90.5 C-terminus does not affect its proper chloroplast localization and dimerization. Finally, transmission electron microscopy indicated that chloroplast development in HSP90.5 cosuppression leaves was significantly impaired and the integrity of chloroplast is highly correlated to the expression level of HSP90.5.ConclusionWe thoroughly characterized three HSP90.5 cosuppression lines, and demonstrated that properly controlled expression of HSP90.5 is required for plant growth and development in many tissues, and especially essential for chloroplast thylakoid formation. Since the homozygote of HSP90.5 knockout mutant is embryonically lethal, this study provides transgenic lines that mimic the conditional knockout line or siRNA line of the essential HSP90.5 gene in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-643) contains supplementary material, which is available to authorized users.

The complete chloroplast DNA sequence of eleven grape cultivars. simultaneous resequencing methodology

<p style="text-align: justify;"><strong>Aims</strong>: The chloroplast DNA sequence of eight Georgian grape cultivars (Rkatsiteli, Saperavi, Meskhuri Mtsvane, Chkhaveri, Aladasturi, Krakhuna, Tsitska, Tsolikouri) and three French cultivars (Chardonnay, Gouais Blanc, Chasselas), belonging to four different haplogroups (AAA, ATT, ATA, GTA), was determined by Illumina resequencing of genomic DNA. The chloroplast DNA sequence of the Maxxa cultivar was used as reference.</p><p style="text-align: justify;"><strong>Methods and results</strong>: The comparison of sequenced chloroplast DNA gave 100 % identity to Chardonnay and Gouais Blanc, differing from Meskhuri Mtsvane by two insertions/deletions (indels) (all ATA haplogroup). The difference between Chasselas and Saperavi was a single insertion (both ATT haplogroup), while Maxxa, Chkhaveri, Aladasturi, Krakhuna, Tsitska and Tsolikouri were all identical (all members of the GTA haplogroup). Forty-seven identical single nucleotide polymorphisms (SNPs) were detected in the AAA, ATA and ATT haplogroups in comparison to the reference DNA. Additionally, 18 SNPs were detected for the ATT haplogroup, 4 for AAA, 6 for ATA and 11 for both AAA and ATA. The phylogenetic results show that the ATT, AAA and ATA haplogroups are more closely related to each other than to the GTA haplogroup.</p><p style="text-align: justify;"><strong>Conclusion</strong>: In the sequencing data of grape genomic DNA at the coverage (read depth) of chromosomal DNA 30-40, the coverage of chloroplast DNA reaches several thousand reads per bp due to the high number of chloroplast DNA copies in genomic DNA, much higher than necessary for resequencing. Based on these data, a new methodology of simultaneous resequencing of large number of chloroplast DNA was developed without preliminary chloroplast isolation or chloroplast enrichment.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: This method has great potential for expanding both phylogenetic and population genetic information on the evolution of domesticated crops.</p>

An improved protocol for intact chloroplasts and cpDNA isolation in conifers.

BackgroundPerforming chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement.Methodology/Principal FindingsWe developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula.ConclusionResults show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.

Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves

This rapid protocol allows the extraction of chloroplast enriched proteins from Nicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined with western-blot analysis. Agrobacterium-mediated transformation (Agroinfiltration, Romeis et al., 2001) is used to transiently express a protein carrying an epitope tag in tobacco leaves. Here, co-infiltration with an Agrobacterium strain harboring 19 K from soil-borne wheat mosaic virus suppresses posttranscriptional gene silencing and therefore increases transformation efficiency (Te et al., 2005).The chloroplast isolation of the transformed leaves is based with modifications on Romeis et al. (2001), and includes mechanical breakage of cell wall and membranes, the removal of unbroken tissue by filtration and the separation of intact chloroplasts by centrifugation through a Percoll layer., 该快速方案允许从瞬时转化以表达感兴趣的表位标记蛋白的本氏烟草（本氏烟草）叶中提取富含叶绿体的蛋白。因此，当它与Western印迹分析结合时，它可以作为研究目标蛋白质的叶绿体定位的工具。使用土壤杆菌介导的转化（Agroinfiltration，Romeis等人，2001）瞬时表达在烟草叶中携带表位标签的蛋白质。在这里，与土壤传播的小麦花叶病毒携带19K的农杆菌菌株的共浸润抑制转录后基因沉默，并因此增加转化效率（Te等人，2005） 。转化的叶子的叶绿体分离基于Romeis等人（2001）的修改，包括细胞壁和膜的机械破坏，通过过滤去除未破坏的组织，通过Percoll层离心分离完整叶绿体。

Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for Illumina genome sequencing

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Kruger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.

Photosynthesis research protocols

This book is number 684 in Springer's popular ‘Methods in Molecular Biology’ series, which is well known for its targeted protocol chapters, each with detailed recipes, photographs, and a set of notes for additional practical guidance. As its name says, Photosynthesis research protocols focuses on various aspects of photosynthesis research and its chapters reflect the whole complexity of this research field. Its editor Robert Carpentier is a very active member of the photosynthesis research community and is involved in three contributions to this book. The first part of the book, with 12 chapters, consists of biochemical protocols. Detailed descriptions of how to purify entire thylakoid multi-protein complexes or parts of them, as well as protocols of their crystallisation, are provided. Moreover, protocols on how to isolate the luminal proteins plastocyanin and cytochrome c6, as well as chloroplast lipids, are provided in this section. The second part of the book with seven chapters is less homogenous and includes protocols on in vitro import of proteins, proteomics, detection of free radicals and reactive oxygen species, photoinhibition assays, reconstitution of PSII into liposomes and a protocol on how to immobilize photosynthetic material. The third part of the book, with four chapters, deals with genetic approaches, focusing on Arabidopsis mutants, cyanobacterial genetics and chloroplast transformation in Chlamydomonas. A small fourth part with only two chapters, the content of which is related to the first biochemical part of this book, presents protocols on chloroplast isolation and the isolation of bundle-sheath cell strands and thylakoids from leaves of C4 grasses. The fifth and final part of the book, with five chapters, describes recipes centred around Rubiso and Rubisco activase, including its isolation, quantitation and activity measurements. All chapters are of high quality and written by leading scientists in their fields. Illustrations are provided when needed and each protocol is sufficiently detailed also to allow access to beginners in the field. The book occasionally gives the impression that the authors were undecided as to whether to focus on photosynthesis or to cover chloroplast biology in general. Hence, this collection of protocols is too diverse to expect that all of it will appeal to every reader. On the other hand, this same diversity ensures that every photosynthesis researcher will surely find several chapters of interest to them within this book.

Isolation of intact and pure chloroplasts from leaves of Arabidopsis thaliana plants acclimated to low irradiance for studies on Rubisco regulation

<p>A protocol is presented for low-cost and fast isolation of intact and pure chloroplasts from leaves of plants acclimated to low irradiance. The protocol is based on a differential centrifugation of cleared leaf homogenate and omits a centrifugation on Percoll gradient step. The intactness and purity of the chloroplasts isolated from leaves of low irradiance-acclimated plants by using this protocol (confirmed by phase contrast microscopy as well as enzymatic and immunological approaches) allows plausible studies on low irradiance-dependent Rubisco regulation.</p>

Isolation of Arabidopsis thylakoid membranes and their use for in vitro protein insertion or transport assays.

This chapter focuses on the techniques of chloroplast isolation; their fractionation into envelopes, stroma, and thylakoids; and their further use for in vitro protein transport assays. In addition to the isolation of thylakoids, this chapter also describes the experimental steps of both protein translocation across the thylakoid membrane and protein integration into the membrane. Protein translocation and integration can be analysed by the radioactive labelling of substrate proteins using an in vitro transcription and translation system. The translocated or integrated proteins can then be detected by autoradiography. Our protocol allows the analysis of these transport systems in wild-type Arabidopsis or mutants that lack or overexpress soluble or membrane transport factors that could be of potential interest.

Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics

The moss Physcomitrella patens is increasingly being used as a model for plant systems biology studies. While genomic and transcriptomic resources are in place, tools and experimental conditions for proteomic studies need to be developed. In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema. Routinely, 60–100 μg mitochondrial and 3–5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue. Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed. This isolation protocol and these validated compartment markers may serve as basis for sub-cellular proteomics in P. patens and other mosses.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-010-0935-4) contains supplementary material, which is available to authorized users.

Chloroplast genome sequences from total DNA for plant identification

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost-effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non-trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost-effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single-locus DNA barcode for plants.

A systemic proteomic analysis of Populus chloroplast by using shotgun method

The chloroplast is one of the most important organelles in plants. Proteomic investigations of chloroplasts have been undertaken for many herb plant species, but to date no such investigation has been reported for woody plant chloroplasts. In the present study we initiated a systematic proteomic study of Populus chloroplasts using a shotgun proteomic method. After isolation of chloroplasts and tryptic digestion of the proteins, the protein fragments were separated via HPLC using an SCX column, and the peptides were analyzed by LC-MS/MS; 119 proteins were successfully identified. Based on annotation information in the UniProtKB/Swiss-Prot database, these proteins were identified as being localized in the chloroplast thylakoid membrane, chloroplast stroma, chloroplast thylakoid lumen, and plastoglobules. Over 50% of all identified proteins were confirmed as chloroplast thylakoid proteins, and 85 are encoded by the chloroplast genome with the remaining proteins encoded by the nuclear genome. Based on functional annotation, these proteins were classified into four functional categories, including photosynthesis, redox regulation and stress, primary and secondary metabolism, transport and signaling. These data provide a valuable basis for further studies on photosynthesis in poplar species.

Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform

BackgroundComplete chloroplast genome sequences provide a valuable source of molecular markers for studies in molecular ecology and evolution of plants. To obtain complete genome sequences, recent studies have made use of the polymerase chain reaction to amplify overlapping fragments from conserved gene loci. However, this approach is time consuming and can be more difficult to implement where gene organisation differs among plants. An alternative approach is to first isolate chloroplasts and then use the capacity of high-throughput sequencing to obtain complete genome sequences. We report our findings from studies of the latter approach, which used a simple chloroplast isolation procedure, multiply-primed rolling circle amplification of chloroplast DNA, Illumina Genome Analyzer II sequencing, and de novo assembly of paired-end sequence reads.ResultsA modified rapid chloroplast isolation protocol was used to obtain plant DNA that was enriched for chloroplast DNA, but nevertheless contained nuclear and mitochondrial DNA. Multiply-primed rolling circle amplification of this mixed template produced sufficient quantities of chloroplast DNA, even when the amount of starting material was small, and improved the template quality for Illumina Genome Analyzer II (hereafter Illumina GAII) sequencing. We demonstrate, using independent samples of karaka (Corynocarpus laevigatus), that there is high fidelity in the sequence obtained from this template. Although less than 20% of our sequenced reads could be mapped to chloroplast genome, it was relatively easy to assemble complete chloroplast genome sequences from the mixture of nuclear, mitochondrial and chloroplast reads.ConclusionsWe report successful whole genome sequencing of chloroplast DNA from karaka, obtained efficiently and with high fidelity.

Simultaneous isolation of intact mitochondria and chloroplasts from a single pulping of plant tissue

Isolated organelles are suitable tools for the investigation of organelle function. However, if the properties of different organelles are to be compared, analysis is generally impeded by the fact that the organelles are isolated independently from each other from different specimens, different tissues or even different plants, i.e. the organelles have been exposed to different conditions during growth and development. Here we describe a method to isolate intact chloroplasts and mitochondria simultaneously from a single pulping of pea leaves, which results in organelles with an essentially identical physiological background. The functionality of the isolated chloroplasts and mitochondria is demonstrated by protein transport experiments, which yield results identical to those obtained with independently isolated organelles. With slight modifications, the method is also successfully applied to organelles from potato and spinach, which implies that it may be generally applicable to organelles from many different species.

Isolating intact chloroplasts from small Arabidopsis samples for proteomic studies

We have established a method for the isolation of chloroplasts from Arabidopsis thaliana that allows proteomic studies in the context of biotic stress with small amounts of starting material. Employing a 50% Percoll layer to separate crude filtrates, the required leaf material was reduced to 2–3 g, yielding more than 300 μg of chloroplast proteins. The quality of this fraction was confirmed by immunological, enzymatic, and gel-based assays. This protocol provides intact chloroplasts from Arabidopsis plants with a high degree of integrity and purity as well as sufficient protein recovery, thereby enabling studies of plant–herbivore or plant–pathogen interactions.