Chloroplast Research Articles

Shallow Whole Genome Sequencing for the Assembly of Complete Chloroplast Genome Sequence of Arachis hypogaea L.

The chloroplast (CP) is a plant organelle originated from cyanobacteria through symbiosis and had become an important component of the plant cell. It is the reaction center for the photosynthesis and also for several steps in the biosynthetic pathways of fatty acids, vitamins, pigments and amino acids. The CP genome is highly conserved in land plants (Raubeson and Jansen, 2005). The CP genome is circular and exhibits a quadripartite genome structure consisting of a large single copy region (LSC) and a small single copy region (SSC), separated by a pair of inverted repeats (IRs) with a few exceptions where loss of an IR or the SSC was observed. The size of the CP genome varies from 19 to 217 Kb in land plants, and the IRs are usually 20–26 kb in length (http://www.ncbi.nlm.nih.gov/genome/organelle/). Lack of recombination makes the CP genome an ideal target for phylogenetic studies (Ravi et al., 2008; Wu and Ge, 2012). Arachis hypogaea L. also known as groundnut is an herbaceous plant belonging to the Fabaceae family. It has an allotetraploid genome (AABB; 2n = 4x = 40) with a size of about 2.8 Gb. There have been many speculations regarding the ancestors of A and B subgenomes of A. hypogaea and proved to have originated through a hybridization event between Arachis ipaensis L. (B subgenome) and Arachis duranensis L. (A subgenome) (Kochert et al., 1996; David et al., 2016). It is one of the major edible oilseed crops in the world, and India is the second largest producer accounting for about 15% of the world production (FAOSTAT, 2015). Kernels of A. hypogaea L. contains 43–50% oil and 23–26% proteins. The oil comprises majorly of palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), arachidic acid (20:0), eicosenoic acid (20:1), behemic acid (22:0), and lignoseric acid (24:0) along with trace amounts of palmitoleic acid (16:1). The mono and poly-unsaturated fatty acids, oleic acid and linoleic acid constitute about 75% of the total oil content (Shiv, 1982). Many attempts have successfully been made to improve the crop yield, drought resistance, disease resistance and other characteristics of A. hypogaea L. using classical breeding as well as genetic engineering using nuclear transformation. Chloroplast transformation by homologous recombination for producing transgenic plants is also possible due to the presence of candidate loci on the CP genome. Additionally, Genetic engineering of chloroplast genome when compared to nuclear transformation is environment-friendly; it minimizes the pleiotropic effects along with containment of the foreign genes (Daniell et al., 2005). Hence, the availability of the complete chloroplast genome of A. hypogaea L. will be an invaluable resource for designing and evaluating efficient chloroplast transformation experiments.

The adaptive significance of differences of root morphology, anatomy and physiology from three ecotypes of reed (Phragmites communis Trin.)

Reeds are widely distributed in drought and high salt conditions of northwestern China. Leaf epidermal micromorphology, anatomy, chloroplast ultrastructure and physio-chemical characteristics due to long-term adaptation in the natural habitats of common reed ( Phragmites communis Trin.) contrasted considerably among three different ecotypes: dune reed (DR), Gobi salt reed (GSR) and swamp reed (SR). The main objective of the present study is to determine the adapting characteristics of morphology, anatomy and physiological responses of thin roots in DR, GSR and SR. The results show that root length density was higher in SR and few root hairs were observed in DR. Cross-section anatomical features show that each ecotype has an endodermis and exodermis, while cortex thickness and proportion of root cortical aerenchyma and stele in root structure varied among the three ecotypes. The stele and xylem share a larger area in DR compared to GSR and SR. GSR has a large proportion of the cortex with radialized distribution of aerenchyma cells spacing, and the cortex has a peripheral, mechanically stiff ring in the exodermis. SEM and TEM microscope images show that GSR has a sclerenchyma ring with high lignification in the exodermis. The physio-chemical parameters show that GSR had a higher level of stress tolerance than DR. These findings indicate that developed water-absorbing tissues were largely distributed in the root structure of DR, and a main framework with supporting function spacing with aerenchyma was dominant in GSR in the long term adaptation to their natural habitats, respectively.

Highly rearranged and size-variable chloroplast genomes in conifer II clade (cupressophytes): Selection toward shorter intergenic spacers

Highly rearranged and size-variable chloroplast genomes in conifer II clade (cupressophytes): Selection toward shorter intergenic spacers

The complete chloroplast genome of Gentiana straminea (Gentianaceae), an endemic species to the Sino-Himalayan subregion

Endemic to the Sino-Himalayan subregion, the medicinal alpine plant Gentiana straminea is a threatened species. The genetic and molecular data about it is deficient. Here we report the complete chloroplast (cp) genome sequence of G. straminea, as the first sequenced member of the family Gentianaceae. The cp genome is 148,991bp in length, including a large single copy (LSC) region of 81,240bp, a small single copy (SSC) region of 17,085bp and a pair of inverted repeats (IRs) of 25,333bp. It contains 112 unique genes, including 78 protein-coding genes, 30 tRNAs and 4 rRNAs. The rps16 gene lacks exon2 between trnK-UUU and trnQ-UUG, which is the first rps16 pseudogene found in the nonparasitic plants of Asterids clade. Sequence analysis revealed the presence of 13 forward repeats, 13 palindrome repeats and 39 simple sequence repeats (SSRs). An entire cp genome comparison study of G. straminea and four other species in Gentianales was carried out. Phylogenetic analyses using maximum likelihood (ML) and maximum parsimony (MP) were performed based on 69 protein-coding genes from 36 species of Asterids. The results strongly supported the position of Gentianaceae as one member of the order Gentianales. The complete chloroplast genome sequence will provide intragenic information for its conservation and contribute to research on the genetic and phylogenetic analyses of Gentianales and Asterids.

Properties and localization of the photoreceptor active in displacements of chloroplasts in Funaria hygroinetrica. II. Studies with polarized light

Properties and localization of the photoreceptor active in displacements of chloroplasts in Funaria hygroinetrica. II. Studies with polarized light

Cloning and characterization of a salt responsive gene AcPsbQ1 from Atriplex canescens.

PsbQ is an extrinsic subunit of the photosystem II in eukaryotic photosynthetic organisms. Numerous studies have demonstrated that PsbQ can stabilize the inorganic cofactors and enhance the oxygen release in PSII. The decrease of photosynthesis rate under salinity condition is normally attributed to the high concentration of injurious ions, such as Na(+) and Cl(-), which accumulate in the chloroplast and damage thylakoid membrane under salinity stress. In this study, AcPsbQ1 was isolated from a halophyte Atriplex canescens cDNA library. The AcPsbQ1 contains an open reading frame of 699 bp encoding a 233 amino acid protein. In order to investigate its function, AcPsbQ1 was cloned and transformed into Saccharomyces cerevisiae INVSc1. The heterologous expression of AcPsbQ1 in transgenic yeast significantly helped to increase the adapting and recovery ability of yeast cells under the salt and drought. Quantitative real-time PCR assay was performed to reveal the expression pattern of AcPsbQ1 under different abiotic stresses. On exposure to NaCl stress, the transcript level of AcPsbQ1 was significantly enhanced. AcPsbQ1 expression level was also up-regulated under drought stress. These results indicated that AcPsbQ1 might involve in the response to salt stress in A. canescens.

A Peculiar Amphitropical Genus of Paniceae (Poaceae, Panicoideae)

Fil: Scataglini, Maria Amalia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Botanica Darwinion. Academia Nacional de Ciencias Exactas, Fisicas y Naturales. Instituto de Botanica Darwinion; Argentina

Characterization of chloroplast matK sequences of Citrus tachibana and Citrus depressa, two indigenous species in Japan

Citrus tachibana, C. nippokoreana, and C. depressa are indigenous mandarin species in Japan. We deduced their phylogenetic relationships from nucleotide sequences of the chloroplast matK gene. The results indicate that C. tachibana, C. nippokoreana, and C. depressa accessions can be classified into two types: type A, all sixteen C. tachibana and six C. Depressa; type B, eleven C. depressa and one C. nippokoreana. Both type A and type B accessions of C. depressa were found on the Okinawa Islands, whereas only type B accessions of C. depressa were found on the Sakishima and Amami Islands. This cpDNA divergence seemed to indicate a polyphyletic origin of C. depressa. The matK genes of type A were found only in C. tachibana and some C. depressa . From these results, both species probably possess a characteristic chloroplast genome among various Citrus species.

Phylogenetic Analysis of the Far Eastern Salix (Salicaceae) Based on Sequence Data from Chloroplast DNA Regions and ITS of Nuclear Ribosomal DNA

In order to further elucidate the relationships in the Salix genus, phylogenetic ana lysis was conducted based on comparison of the nucleotide sequences of the chlo roplast DNA regions (petG–trnP, petN–psbM, psaA–ycf3, psbM–trnD, rpoB– trnC, trnC–petN, trnD–trnT, trnS–psbZ, rbcL, matK) and the ITS region (ITS1–5.8S rRNA–ITS2) of nuclear ribosomal DNA of 86 Salix species from three sub­ genera: Salix, Chamaetia, Vetrix, 27 sections and Chosenia arbutifolia. The previous ly recognized genera Chosenia and Toisusu merged with Salix. The divergence of spe­ cies in the Salix genus (according to ITS and cpDNA data) was very low. In most spe cies from Chamaetia and Vetrix subgenera, the sequences differed by several sub­stitutions­or­were­ identical.­Molecular­differences­were­ identified­mainly­ in­ spe cies belonging to the subgenus Salix in its previous broad meaning. ITS­phy­ lo­geny­generally­agrees­with­the­most-recent­infrageneric­classification­of ­Salix: the isolation of subgenera Pleuradenia, Chosenia, Salix (for exception sect. Triand­ rae), Longifoliae has been well supported, but the isolation of subgenera Protitea and Chamaetia is not supported. Relationships between the subgenera Chamaetia and Vetrix remain unresolved. High similarity of the plastid and nuclear genomes re vealed in the majority of Salix species included in the analysis may indicate that they have relatively recently diverged from a common ancestor or there is a result of lineage sorting through hybridization. K e y w o r d s chloroplast DNA, ITS, phylogenetic analysis, Salicaceae, Salix

April 2014: This Month in JoVE - Bioengineering for ACL Tears, Neuroscience behind Insect Locomotion, Improved Genetic Engineering of Crops, and Synthesizing Secret Ink

Here's a look at what's coming up in the April 2014 issue of JoVE: The Journal of Visualized Experiments. In the Bioengineering section we have a video article that examines the anterior cruciate ligament (ACL), which is critical for keeping the knee stable when pivoting or planting. Torn or ruptured ACLs are among the most common of all serious knee injuries, especially in athletes, and even partial tears can hurt knee stability and function. Because they can't heal on their own, torn ACLs are often treated surgically, sometimes by reconstructing the ligament with a tendon grafted from the patient's own body or from a cadaver. Every year, over 100,000 ACL reconstructions are performed in the United States. The torn ligaments are usually discarded as surgical waste, but Gupta et al. see them as a valuable source of cells for potentially repairing ACLs. They developed a method for harvesting and expanding human ACL-derived cells then growing them on scaffolds to form tissue-engineered patches. Under scanning electron microscopy, the engineered matrix demonstrates good cellular adherence and growth. This technique could lead to methods for strengthening damaged ligaments and enhancing ACL repair, especially for those wishing to maintain an athletic lifestyle. In the Neuroscience section we examine motor control in insects, which are among the most athletic creatures on Earth. They are especially good at adapting to sudden changes in terrain, so scientists are interested in correlating neural activity with behavior in insects. Guo et al. have developed a method for recording brain activity in the discoid cockroach, also known as the tropical cockroach, as they explore an arena or as they encounter obstacles that cause them to change course. The authors also demonstrate how to collect the data for subsequent analysis, which may yield insight into the neural control of behavior. In JoVE Environment we feature a technique for transient gene expression in plants. To express gene products in specific parts of the cell, plants often have several copies of a gene and express them separately in different organelles (such as the chloroplast and peroxisome) or in the cytosol. Therefore, genetic engineers may have to target multiple duplicated genes to manipulate expression in different cell parts. To get around this problem, Mattozzi et al. use a novel method for multiple-organelle targeting that takes advantage of alternatively spliced mRNAs, which are sent to different locations in the cell. The targeting constructs are designed using Gibson assembly, a one-step cloning method, and then delivered using a gene gun. This technique offers great flexibility in terms of genetic design, and can be applied to large-scale selective genetic engineering in agriculture or the environment. In the Chemistry section we feature an article that uses modern chemical techniques to examine two bright blue pigments that were synthesized in ancient societies. Calcium copper tetrasilicate, commonly known as Egyptian blue, is thought to be the first synthetic pigment and used in Egypt as early the 4th Dynasty (ca. 2500 BC), the height of the pyramid-building era. Barium copper tetrasilicate, also known as Han blue, was later developed in China during the beginning of the Zhou Dynasty, or the height of the bronze age (1045-771 BC). Nowadays, scientists are interested in the strong near infrared (NIR) emission properties of these two pigments, which can be utilized for optical sensors. Related to this application is the ability to exfoliate copper tetrasilicates into 2D monolayers, or nanosheets. Johnson-McDaniel and Salguero demonstrate how to synthesize these two copper tetrasilicate pigments and exfoliate them into monolayer nanosheets. Egyptian blue spontaneously exfoliates into monolayers when stirred in hot water, whereas Han blue requires ultrasonication in organic solvents. Our authors show that both the bulk and nanosheet forms of the two pigments are strong near infrared emitters with potential applications ranging from security inks to biomedical imaging. You've just had a sneak peek of the April 2014 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Molecular and physiological role of Epipremnum aureum

Epipremnum aureum (Golden pothos) is a naturally variegated climbing vine that produces abundant yellow‑marbled foliage. It is among the most popular tropical ornamental plant used as hanging basket crop. An insight has been provided about the different varieties of Golden pothos including Neon, Marble Queen, Jade Pothos and N Joy. This paper presents a critical review on botanical study and important characteristics of Golden pothos and special emphasis has been provided on variegated leaves and chloroplast biogenesis explaining the important genes involved during the process and various proteins associated with it. Studies have been included comprising the special features of Epipremnum aureum in phytoremediation for the removal of Cobalt and Cesium and in the purification of air against formaldehyde. The antimicrobial activity of roots and leaf extracts of Epipremnum aureum against many bacterial strains have been included. It also presents the antitermite activity of Golden pothos that can be harnessed for pest control. Key words: Antimicrobial, antitermite, calcium oxalate, Epipremnum aureum, formaldehyde

DNA extraction and molecular identification of imported Dalbergia wood

Establishing a reliable and cost effective method to identify and distinguish wood is important in preventing the illegal timber trade. To overcome the difficulty of distinguishing Dalbergia，which consists of many valuable species，by morphological methods，molecular genetic tools to control species identity were studied. Seven species of Dalbergia wood，D. melanaoxylon，D. oliveri，D. retusa，D. louvelii，D. greveana，D. cochinchinensis，D. cultrate，imported from different countries and one domestic species D. odorifera were selected for testing. Next，an array of different wood DNA extraction and purification methods，including DNeasy Plant Mini Kit，hexadecyltrimethylammonium bromide（CTAB），sodium dodecyl sulfate （SDS） and Magnetic Beads based method，were tried；For the amplification of DNA from wood samples, two PCR reactions were successively performed（nested PCR）and allowed the amplication of one coding region of chloroplast DNA （cpDNA）. Results showed that a CTAB-SDS-Magnetic Beads based method was available for Dalbergia wood DNA extraction. Then，433 bp matK gene fragments were amplified and sequenced from the seven species，of which a total of 12 single nucleotide polymorphisms were found. These polymorphic sites were able to distinguish the eight Dalbergia species. Thus，establishment of this method for DNA isolation and polymerase chain reaction （PCR） amplification makes Dalbergia wood amenable to DNA-based identification.［Ch，2 fig. 1 tab. 23 ref.］

THE EYESPOT MEMBRANES OF HAEMATOCOCCUS PLUVIALIS FLOTOW (CHLOROPHYCEAE): THEIR ULTRASTRUCTURE AND POSSIBLE SIGNIFICANCE IN PHOTOTAXIS

SUMMARY The eyespot region of the green flagellate alga Haematococcus pluvialis Flotow (Clorophyceae) was investigated by electron microscopy in order to identify any possible specialization of the plasmalemma and the outer chloroplast envelope membrane connected with photoreception. In this alga, the distance between the outer chloroplast membrane and the plasmalemma in the eyespot region is variable. In some thin sections in this region, the inner limiting membrane of the chloroplast is more electron dense than the outer, while the thickness of the plasmalemma opposite the eyespot shows no appreciable difference with respect to other parts of the cell. The possible functional significance of these findings and the possibility that the eyespot in Haematococcus pluvialis serves as a shading device are both discussed.

DIRECT DEVELOPMENT AND SYMBIOTIC CHLOROPLASTS IN ELYSIA TIMIDA (MOLLUSCA: OPISTHO BRANCHI A)

ABSTRACT Elysia timida represents in its development an evolutionarily advanced type, as compared to other described Elysia. Egg number per egg mass is lowest, veliger stage and metamorphosis are completed inside the egg capsule, and a bilateral benthic juvenile hatches after 16–21 days. Mature E. timida contain symbiotic chloroplasts, which persist for two months and are then slowly bleached. Eggs and juveniles are aposymbiotic. Survival time of mature Elysia and juveniles in our cultures was four months and four weeks, respectively. Attempts to induce feeding on various algae, in juveniles or mature Elysia, were unsuccessful. Algal source of the symbiotic chloroplasts in E. timida and the mode of their acquisition remain unknown.

DEVELOPMENT OF CHLOROPLASTS AT HIGH AND LOW LIGHT QUANTA FLUENCE RATES

ABSTRACT Chloroplast ultrastructure, ratio of photosynthetic pigments and the chlorophyll fluorescence induction kinetics (Kautsky effect) of plants grown under high light (HL) and low light (LL) intensity and of sun and shade leaves were compared in order to find parameters which describe the differences between sun-type chloroplasts (HL-chloroplasts) and shade-type ones (LL-chloroplasts). The differences in ultrastructure between HL- and LL-chloroplasts can be quantified by measuring several parameters. As compared to LL-chloroplasts the HL-chloroplasts exhibit a lower frequency of thylakoids (in most plants), a higher proportion of exposed membranes, a lower stacking degree and lower values for the ratio between appressed to exposed membranes, a narrower grana width, fewer thylakoids per granum, a high starch content and a greater number and larger size of plastoglobuli. As compared to LL-chloroplasts, HL-chloroplasts are characterized by a higher chlorophyll a and (β-carotene content, which results in...

INVESTIGATION OF CHLOROPLAST MEMBRANE FORMATION AND ITS CONTROL WITH THE AID OF HEAT-BLEACHED 70S RIBOSOME-DEFICIENT LEAVES

ABSTRACT The formation of pigments, lipids and proteins of the chloroplast membranes in heat-treated 70S ribosome-deficient leaves is reviewed. In the light, 70S ribosome-deficient leaves were chlorotic, although they must contain virtually all the enzymes of chlorophyll biosynthesis. Carotenoids and all major chloroplast lipids (glycolipids mono-galactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol and prenylquinones plastoquinone-9, α-tocopherol, phylloquinone) were formed in heat-bleached 70S ribosome-deficient leaves. Their amounts were, however, lower than in normal green leaves because their synthesis appeared to be coupled by regulatory mechanisms to the availability of chlorophyll. Several membrane proteins and functions of the plastid envelope (e.g. dicarboxylic acid translocator activity) and internal membranes (e.g. ferredoxin, ferredoxin-NADP reductase, light-harvesting chlorophyll a/b- protein) were identified in 70S ribosome-deficient leaves. The 69 kD apoprotei...

CHLOROPLAST DEVELOPMENT IN ZEA MAYS

C4 plants differ from C3 plants in leaf anatomy in that two distinct cell types cooperate to carry out photosynthesis and in that these cells possess structurally and functionally dimorphic chloroplasts. The situation is further complicated by the occurrence of three distinct varieties of C4 photosynthesis. The state of knowledge with respect to chloroplast development in the most investigated C4 species, Zea mays, is reviewed. Fine structural dimorphism is relatively easy to investigate while the development of functional dimorphism is more difficult to understand. It is, however, an important fundamental question as to how nuclear and plastid genomes are expressed differently in adjacent cells so as to produce this dimorphism.

REGULATION AND EXPRESSION OF GENES FOR CHLOROPLAST PROTEINS

Abstract The biosynthesis and function of a chloroplast are controlled by genes located in two genomes, that in the nucleus of the photosynthetic cell and that in the organelle itself. This dual location raises many questions as to how the nuclear and chi genes which code chl-specific ribonucleic acids and proteins are regulated and coordinated to produce a functioning organelle. Though much remains to be answered, the overall pattern of expression of genes coding chi proteins is becoming clear. This review considers the expression of the genes coding two chi proteins, i.e. the light-harvesting chlorophyll a/b protein which is coded in the nucleus, and the enzyme ribulose-1,5- bisphosphate carboxylase which is composed of two subunits, one of which is coded in the nucleus and the other in the chloroplast. All levels of expression of these genes are considered from their structure and transcription to the final assembly and integration of their functional protein products into the appropriate compartment o...

ULTRASTRUCTURE OF PLASTIDS OF PARASITIC HIGHER PLANTS. V. INFLUENCE OF LIGHT ON CUSCUTA PLASTIDS

Abstract The ultrastructure of plastids in light-grown stems of Cuscuta australis is examined. The ultrastructure of the plastids is greatly altered if the stems are placed in darkness. Under these conditions chloroplasts with grana formed by several thylakoids connected by stroma lamellae are found, while in illuminated stems the plastids show characters of amyloplasts. The chlorophyll content and the photosynthetic activity sometimes observed in Cuscuta are probably correlated with the fact that this plant is able to build the ultrastructure of the photosynthetic apparatus only in weak light or in darkness.

GENETIC CHANGES INDUCED IN HIGHER PLANTS BY A UV LASER MICROBEAM

Plant cell walls can be perforated by a UV laser microbeam. Cells and immature pollen grains (microspores) of Brassica napus L. continued to grow in culture after being punctured by a laser pulse. During laser treatment, uptake of buffer into the cytoplasm of cells could be achieved by plasmolyzing cells in a hypertonic solution. Using this approach, plasmid DNA carrying a gene for bacterial glucuronidase was introduced into B. napus cells. The marker gene was expressed by 70% of the treated cells. Likewise, the gene for resistance to the antibiotic hygromycin B was introduced into B. napus cells and was stably integrated into growing cells. Furthermore, DNA was also incorporated into chloroplasts. First, DNA was injected into the cytoplasm and then the organelles were punctured by laser pulses. When a marker gene coding for triazine resistance was introduced into the organelles, transient expression at the level of callus cultures was observed.