Chloroplast Research Articles

Comparative Analysis and Phylogeny of the Complete Chloroplast Genomes of Nine Cynanchum (Apocynaceae) Species.

Cynanchum belongs to the Apocynaceae family and is a morphologically diverse genus that includes around 200 shrub or perennial herb species. Despite the utilization of CPGs, few molecular phylogenetic studies have endeavored to elucidate infrafamilial relationships within Cynanchum through extensive taxon sampling. In this research, we constructed a phylogeny and estimated divergence time based on the chloroplast genomes (CPGs) of nine Cynanchum species. We sequenced and annotated nine chloroplast (CP) genomes in this study. The comparative analysis of these genomes from these Cynanchum species revealed a typical quadripartite structure, with a total sequence length ranging from 158,283 to 161,241 base pairs (bp). The CP genome (CPG) was highly conserved and moderately differentiated. Through annotation, we identified a total of 129-132 genes. Analysis of the boundaries of inverted repeat (IR) regions showed consistent positioning: the rps19 gene was located in the IRb region, varying from 46 to 50 bp. IRb/SSC junctions were located between the trnN and ndhF genes. We did not detect major expansions or contractions in the IR region or rearrangements or insertions in the CPGs of the nine Cynanchum species. The results of SSR analysis revealed a variation in the number of SSRs, ranging from 112 to 150. In five types of SSRs, the largest number was mononucleotide repeats, and the smallest number was hexanucleotide repeats. The number of long repeats in the cp genomes of nine Cynanchum species was from 35 to 80. In nine species of Cynanchum, the GC3s values ranged from 26.80% to 27.00%, indicating a strong bias towards A/U-ending codons. Comparative analyses revealed four hotspot regions in the CPG, ndhA-ndhH, trnI-GAU-rrn16, psbI-trnS-GCU, and rps7-ndhB, which could potentially serve as molecular markers. In addition, phylogenetic tree construction based on the CPG indicated that the nine Cynanchum species formed a monophyletic group. Molecular dating suggested that Cynanchum diverged from its sister genus approximately 18.87 million years ago (Mya) and species diversification within the Cynanchum species primarily occurred during the recent Miocene epoch. The divergence time estimation presented in this study will facilitate future research on Cynanchum, aid in species differentiation, and facilitate diverse investigations into this economically and ecologically important genus.

Characterization, comparison, and phylogenetic analyses of chloroplast genomes of Euphorbia species

The genus Euphorbia (Euphorbiaceae) has near-cosmopolitan distribution and serves as a significant resource for both ornamental and medicinal purposes. Despite its economic importance, Euphorbia's taxonomy has long been challenged by the intricate nature of morphological traits exhibiting high levels of convergence. While molecular markers are essential for phylogenetic studies, their availability for Euphorbia has been limited. To address this gap, we conducted comparative analyses focusing on the chloroplast (CP) genomes of nine Euphorbia species, incorporating three newly sequenced and annotated accessions. In addition, phylogenetic informativeness and nucleotide diversity were computed to identify candidate markers for phylogenetic analyses among closely related taxa in the genus. Our investigation revealed relatively conserved sizes and structures of CP genomes across the studied species, with notable interspecific variations observed primarily in non-coding regions and IR/SC borders. By leveraging phylogenetic informativeness and nucleotide diversity, we identified rpoB gene as the optimal candidate for species delimitation and shallow-level phylogenetic inference within the genus. Through this comprehensive analysis of CP genomes across multiple taxa, our study sheds light on the evolutionary dynamics and taxonomic intricacies of Euphorbia, offering valuable insights into its CP genome evolution and taxonomy.

Membrane Fluidization Governs the Coordinated Heat-Inducible Expression of Nucleus- and Plastid Genome-Encoded Heat Shock Protein 70 Genes in the Marine Red Alga Neopyropia yezoensis.

Heat shock protein 70 (HSP70) is an evolutionarily conserved protein chaperone in prokaryotic and eukaryotic organisms. This family is involved in the maintenance of physiological homeostasis by ensuring the proper folding and refolding of proteins. The HSP70 family in terrestrial plants can be divided into cytoplasm, endoplasmic reticulum (ER)-, mitochondrion (MT)-, and chloroplast (CP)-localized HSP70 subfamilies. In the marine red alga Neopyropia yezoensis, the heat-inducible expression of two cytoplasmic HSP70 genes has been characterized; however, little is known about the presence of other HSP70 subfamilies and their expression profiles under heat stress conditions. Here, we identified genes encoding one MT and two ER HSP70 proteins and confirmed their heat-inducible expression at 25 °C. In addition, we determined that membrane fluidization directs gene expression for the ER-, MT-, and CP-localized HSP70 proteins as with cytoplasmic HSP70s. The gene for the CP-localized HSP70 is carried by the chloroplast genome; thus, our results indicate that membrane fluidization is a trigger for the coordinated heat-driven induction of HSP70 genes harbored by the nuclear and plastid genomes in N. yezoensis. We propose this mechanism as a unique regulatory system common in the Bangiales, in which the CP-localized HSP70 is usually encoded in the chloroplast genome.

Novel ZIF-8@CHs catalysts for photocatalytic degradation of tetracycline hydrochloride

A novel ZIF-8@CHs photocatalyst, which was generated using the metal organic framework ZIF-8 and intact chloroplasts (CHs) from Chlorella sp. in a one-pot synthesis, was used in this study for the first time to degrade tetracycline hydrochloride (TCH) through photocatalysis. The results of systematic characterization demonstrated that ZIF-8@CHs was successfully p0repared. Meanwhile, ZIF-8@CHs exhibited excellent photocatalytic performance and good adaptability. Under the condition of simulated sunlight, the high porosity ZIF-8 shell enabled CHs to maintain typical photosynthetic oxygen evolution behavior in vitro, and using CHs as the core produced significant ROS, significantly improving the efficiency of TCH photocatalytic degradation. In addition, the radical trapping test and EPR spectra proved that O2–, 1O2, and •OH were the key active species for TCH degradation. Eventually, the ZIF-8@CHs system can not only expand the new concept of organic pollutant degradation in water bodies, but also offer recommendations for live organ envelope modification..

DETECTION OF GENETICALLY MODIFIED RICE LOCALLY AVAILABLE IN PUNJAB, PAKISTAN

Rice (Oryza sativa L.) is an important cereal crop that provides food to half of the world's population. Pakistan's traditional and premium quality rice is mostly exported to Europe. Transgenic or genetically modified rice (GM RICE) has been developed in the USA, China, and other countries, like herbicide-tolerant Liberty-Link rice (LLRICE06, LLRICE62, LLRICE601) and insect-resistant Bt-63. International and national biosafety regulations allow countries to stop the import of any genetically modified organism (GMO) due to its possible safety concerns. PCR-based methods are recommended for the accurate detection of GM rice. Qualitative PCR is used to detect the presence or absence of GMOs in a rice sample. The objective of this study was to check the presence of GMOs in the rice samples collected from rice-growing areas in Punjab. Eighty-two rice samples were collected randomly from various markets/locations in rice-growing areas of Punjab for this study. Twelve bulks were prepared to represent eighty-two rice samples. The CTAB method was used to extract the DNA from rice samples to analyze it. PCR of rice bulk samples was performed using primers specific for SPS, CP, p35S, tNOS, nptII, Hpt and Bt 63 rice. Positive and negative controls were included in each PCR. Primers specific for chloroplast (CP) and Sucrose phosphate synthase (SPS) genes were used to confirm DNA quality for amplification. Then, primers specific for the cauliflower mosaic virus (CaMV) 35S promoter and the Agrobacterium nopaline synthase (NOS) terminator were used for the detection of GM rice through PCR, neomycin phosphotransferase II (nptII) and Hygromycin phosphotransferase (Hpt) were used as a selectable marker and Bt 63 rice was used in insect resistance. The gel electrophoresis of PCR was conducted using a DNA ladder and then taking of the image was through gel documentation system for visualization and analysis. According to the results, all the rice bulks representing the eighty-two rice samples were negative for p35S, tNOS, nptII, Hygro and Bt 63 rice event. These findings are encouraging and demonstrate that rice grown in kalar track of Punjab, Pakistan, is non-GMO. Such surveillance and monitoring would be largely beneficial to maintain the purity of our traditional non-GM rice and to avoid any possible mixing of GM rice seeds, especially at the time of import. All these efforts will ensure and protect our national share in the international trade of rice.

Comparative and phylogenetic analyses of the chloroplast genome reveal the taxonomy of the Morus genus.

Mulberry (genus Morus) is an economically important woody plant with an altered ploidy level. The variable number of Morus species recognized by different studies indicates that the genus is in need of revision. In this study, the chloroplast (CP) genomes of 123 Morus varieties were de novo assembled and systematically analyzed. The 123 varieties represented six Morus species, namely, Morus alba, Morus nigra, Morus notabilis, Morus rubra, Morus celtidifolia, and Morus serrata. The Morus CP genome was found to be 158,969~159,548 bp in size with 125 genes, including 81 protein coding, 36 tRNA, and 8 rRNA genes. The 87 out of 123 mulberry accessions were assigned to 14 diverse groups with identical CP genome, which indicated that they are maternally inherited and share 14 common ancestors. Then 50 diverse CP genomes occurred in 123 mulberry accessions for further study. The CP genomes of the Morus genus with a quadripartite structure have two inverted repeat (IR) regions (25,654~25,702 bp) dividing the circular genome into a large single-copy (LSC) region (87,873~88,243 bp) and small single-copy (SSC) region (19,740~19,994 bp). Analysis of the phylogenetic tree constructed using the complete CP genome sequences of Morus revealed a monophyletic genus and that M. alba consisted of two clades, M. alba var. alba and M. alba var. multicaulis. The Japanese cultivated germplasms were derived from M. alba var. multicaulis. We propose that the Morus genus be classified into six species, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. rubra, and M. alba with two subspecies, M. alba var. alba and M. alba var. multicaulis. Our findings provide a valuable resource for the classification, domestication, and breeding improvement of mulberry.

Formin protein DRT1 affects gross morphology and chloroplast relocation in rice.

Plant height and tiller number are two major factors determining plant architecture and yield. However, in rice (Oryza sativa), the regulatory mechanism of plant architecture remains to be elucidated. Here, we reported a recessive rice mutant presenting dwarf and reduced tillering phenotypes (drt1). Map-based cloning revealed that the phenotypes are caused by a single point mutation in DRT1, which encodes the Class I formin protein O. sativa formin homolog 13 (OsFH13), binds with F-actin, and promotes actin polymerization for microfilament organization. DRT1 protein localized on the plasma membrane (PM) and chloroplast (CP) outer envelope. DRT1 interacted with rice phototropin 2 (OsPHOT2), and the interaction was interrupted in drt1. Upon blue light stimulus, PM localized DRT1 and OsPHOT2 were translocated onto the CP membrane. Moreover, deficiency of DRT1 reduced OsPHOT2 internalization and OsPHOT2-mediated CP relocation. Our study suggests that rice formin protein DRT1/OsFH13 is necessary for plant morphology and CP relocation by modulating the actin-associated cytoskeleton network.

Characterization of complete chloroplast genome in <i>Firmiana kwangsiensis </i>and <i>F. danxiaensis</i> with extremely small populations

Characterization of complete chloroplast genome in <i>Firmiana kwangsiensis </i>and <i>F. danxiaensis</i> with extremely small populations

Characteristic and Phylogenetic Analysis of the Complete Chloroplast Genomes of Three Medicinal Plants of Schisandraceae.

Schisandra chinensis, which has a high development value, has long been used as medicine. Its mature fruits (called Wuweizi in Chinese) have long been used in the famous traditional Chinese medicine (TCM) recorded in the “Chinese Pharmacopoeia.” Chloroplasts (CP) are the highly conserved primitive organelles in plants, which can serve as the foundation for plant classification and identification. This study introduced the structures of the CP genomes of three Schisandraceae species and analyzed their phylogenetic relationships. Comparative analyses on the three complete chloroplast genomes can provide us with useful knowledge to identify the three plants. In this study, approximately 5 g fresh leaves were harvested for chloroplast DNA isolation according to the improved extraction method. A total of three chloroplast DNAs were extracted. Afterwards, the chloroplast genomes were reconstructed using denovo combined with reference-guided assemblies. General characteristics of the chloroplast genome and genome comparison with three Schisandraceae species was analyzed by corresponding software. The total sizes of complete chloroplast genomes of S. chinensis, S. sphenanthera, and Kadsura coccinea were 146875 bp, 146842 bp, and 145399 bp, respectively. Altogether, 124 genes were annotated, including 82 protein-coding genes, 34 tRNAs, and 8 rRNAs of all 3 species. In SSR analysis, only S. chinensis was annotated to hexanucleotides. Moreover, comparative analysis of chloroplast Schisandraceae genome sequences revealed that the gene order and gene content were slightly different among Schisandraceae species. Finally, phylogenetic trees were reconstructed, based on the genome-wide SNPs of 38 species. The method can be used to identify and differentially analyze Schisandraceae plants and offer useful information for phylogenetics as well as further studies on traditional medicinal plants.

Chloroplasts in C3 grasses move in response to blue-light

Key messageBrachypodium distachyonis a good model for studying chloropla st movements in the crop plants, wheat, rye and barley. The movements are activated only by blue light, similar to Arabidopsis.Chloroplast translocations are ubiquitous in photosynthetic organisms. On the one hand, they serve to optimize energy capture under limiting light, on the other hand, they minimize potential photodamage to the photosynthetic apparatus in excess light. In higher plants chloroplast movements are mediated by phototropins (phots), blue light receptors that also control other light acclimation responses. So far, Arabidopsis thaliana has been the main model for studying the mechanism of blue light signaling to chloroplast translocations in terrestrial plants. Here, we propose Brachypodium distachyon as a model in research into chloroplast movements in C3 cereals. Brachypodium chloroplasts respond to light in a similar way to those in Arabidopsis. The amino acid sequence of Brachypodium PHOT1 is 79.3% identical, and that of PHOT2 is 73.6% identical to the sequence of the corresponding phototropin in Arabidopsis. Both phototropin1 and 2 are expressed in Brachypodium, as shown using quantitative real-time PCR. Intriguingly, the light-expression pattern of BradiPHOT1 and BradiPHOT2 is the opposite of that for Arabidopsis phototropins, suggesting potential unique light signaling in C3 grasses. To investigate if Brachypodium is a good model for studying grass chloroplast movements we analyzed these movements in the leaves of three C3 crop grasses, namely wheat, rye and barley. Similarly to Brachypodium, chloroplasts only respond to blue light in all these species.

Impact of drought stress on physiological characteristics and isolation of chloroplasts in common bean (Phaseolus vulgaris L.)

Chloroplasts are involved in many cellular processes and play an important role in plant stress response. In this study, the effect of drought on different physiological characteristics and the isolation of chloroplasts from leaves of drought-stressed and control plants of common bean (Phaseolus vulgaris L.) are reported. Two cultivars differing in the response to drought were analysed-Tiber and more sensitive Starozagorski eern. The results of relative water content showed that plants were under mild stress conditions after six days without watering. Negative effects of drought stress on the photosynthetic rate and chlorophyll fluorescence parameters of both cultivars were observed. The yield of isolated chloroplasts in control samples of both cultivars was at least three times higher compared to drought-stressed samples. The intactness of the isolated chloroplasts was checked and used for protein isolation. The results of the study suggested that even mild drought stress strongly affected the isolation of intact chloroplasts. Still, intact chloroplasts were enriched and were suitable for downstream proteomic analysis.

Enhanced Photogenerated Electron Transfer in a Semiartificial Photosynthesis System Based on Highly Dispersed Titanium Oxide Nanoparticles.

In this study, a hybrid semiartificial photosynthesis system based on chloroplast (CLP) and titanium oxide nanoparticles (TiO2 NPs) was constructed. 2,6-Dichlorophenolindophenol (DCPIP) reduction by TiO2/CLP complex and methylene blue (MB) reduction by TiO2 were used to determine enhanced photogenerated electron transfer in this hybrid system. The DCPIP reduction by the TiO2/CLP complex showed the same trend as MB reduction by TiO2 as a function of concentration of TiO2 NPs, indicating interception of photogenerated electrons in TiO2 by CLP that leads to enhanced photosynthesis efficiency. Decreased photoluminescence intensity and shortened excited-state lifetime of the TiO2/CLP complex compared to that of pure TiO2 also support electron transfer from TiO2 to CLP. Longer visible light absorption wavelength and increasing valence band edges reveal the narrower band gap of TiO2/CLP, which finally results in the enhanced electron transfer from TiO2 to CLP. Higher ferricyanide reduction and enhanced ATP formation with the TiO2/CLP complex demonstrate the accelerated electron-transfer rate of the electron-transfer chain. This study reveals the mechanism of how TiO2 interacts with CLP to enhance the photosynthesis via constructing a semiartificial photosynthesis system.

The complete plastome sequence of Clematis guniuensis (Ranunculaceae), a new plant species endemic to China

Clematis guniuensis is a new plant species in China, which distributes in both provinces of Zhejiang and Anhui with extremely small population. The whole chloroplast (CP) genome was assembled by NovoPlasty using high-throughput sequencing data generated by an Illumina Hiseq X Ten platform. The circular chloroplast genome is 159,682 bp in length, and comprises a large single copy of 79,461 bp, a small single copy of 49,156 bp, and two inverted repeats of 31,064 bp each. The CP genome contains 137 genes, including 89 protein-coding genes, eight rRNA genes, 36 tRNA genes, and four pseudogenes. All the pseudogenes were infA, rpl32, ycf1, and ndhK. Phylogenetic analysis results indicated that C. guniuensis was sister to C. trichotoma, with a support rate of 100%.

Characterization of the complete plastome sequence of Torreya grandis var. jiulongshanensis (Taxaceae), a rare and endangered plant species endemic to Zhejiang province, China

Torreya grandis var. jiulongshanensis (Taxaceae) is an evergreen tree endemic to Zhejiang province, China, and it is listed as a rare plant species with extremely small populations. In our present study, we assembled the chloroplast (CP) genome of T. grandis var. jiulongshanensis using high-throughput sequencing data generated by an Illumina Hiseq X Ten platform. The results indicate that the complete CP genome is 136,705 bp in size with an overall guanine and cytosine content of 35.5%. The plastome has lost its quadripartite structure. In the CP genome, there are a total of 119 genes, including 82 protein-coding genes, 4 ribosomal RNA genes, and 33 transfer RNA genes. Phylogenetic analysis revealed that T. grandis var. jiulongshanensis is a sister species to T. grandis.

CrABCA2 Facilitates Triacylglycerol Accumulation in Chlamydomonas reinhardtii under Nitrogen Starvation.

The microalga Chlamydomonas reinhardtii accumulates triacylglycerols (TAGs) in lipid droplets under stress conditions, such as nitrogen starvation. TAG biosynthesis occurs mainly at the endoplasmic reticulum (ER) and requires fatty acid (FA) substrates supplied from chloroplasts. How FAs are transferred from chloroplast to ER in microalgae was unknown. We previously reported that an Arabidopsis thaliana ATP-binding cassette (ABC) transporter, AtABCA9, facilitates FA transport at the ER during seed development. Here we identified a gene homologous to AtABCA9 in the C. reinhardtii genome, which we named CrABCA2. Under nitrogen deprivation conditions, CrABCA2 expression was upregulated, and the CrABCA2 protein level also increased. CrABCA2 knockdown lines accumulated less TAGs and CrABCA2 overexpression lines accumulated more TAGs than their untransformed parental lines. Transmission electron microscopy showed that CrABCA2 was localized in swollen ER. These results suggest that CrABCA2 transports substrates for TAG biosynthesis to the ER during nitrogen starvation. Our study provides a potential tool for increasing lipid production in microalgae.

Endowing chloroplasts with artificial "cell walls" using metal-organic frameworks.

Biological photosynthesis via chloroplasts (CHs) is widely recognized as the most appropriate and effective method to convert solar energy and simultaneously supply nutrition to maintain life on earth. It is of great significance to prepare CH-based biohybrids which not only can artificially simulate the photocatalytic functionality of CH-containing plants and bacteria, but also can be easily prepared, stored for a long period and conveniently utilized when needed. In this work, for the first time, CHs were encapsulated into metal-organic frameworks (MOFs), namely zeolitic imidazolate frameworks (ZIF-8), under very gentle reaction conditions, i.e. in aqueous solution and at room temperature. Without the negative effects of organic solvents and high temperature on synthesis, the obtained CH@ZIF-8 biohybrids not only have shells maintaining the porous structure of ZIF-8, but also well preserve the biological activity of CHs inside. The porous ZIF-8 coating on CHs acts as a "cell wall" to allow mass and energy exchange between CHs and the environment, and protect CHs from microbiological degradation, which significantly prolong the lifetime of CHs in vitro (raised from several days to >300 days). The CH@ZIF-8 biohybrids may have promising applications in "living" artificial leaves and even artificial trees capable of photosynthesis in the future.

The complete chloroplast genome sequence of Cerasus jingningensis (Rosaceae), an endemic plant to Zhejiang province, China

Cerasus jingningensis is a deciduous shrub endemic to Zhejiang province, China. In our study, we assembled and characterised the chloroplast (CP) genome of C. jingningensis based on high-throughput Illumina sequencing data. The CP genome is 157,913 bp in length with an overall GC content of 36.7%, and it consists of two inverted repeats of 26,427 bp each, a large single copy of 85,934 bp, and a small single copy of 19,125 bp. The genome harbours 131 genes, including 85 protein-coding genes, 37 tRNA genes, eight rRNA genes, and a pseudogene ycf1. Phylogenetic analysis revealed that C. jingningensis is a sister of C. serrulata var. spontanea.

Sequencing and Structural Analysis of the Complete Chloroplast Genome of the Medicinal Plant Lycium chinense Mill.

Lycium chinense Mill, an important Chinese herbal medicine, is widely used as a dietary supplement and food. Here the chloroplast (CP) genome of L. chinense was sequenced and analyzed, revealing a size of 155,756 bp and with a 37.8% GC content. The L. chinense CP genome comprises a large single copy region (LSC) of 86,595 bp and a small single copy region (SSC) of 18,209 bp, and two inverted repeat regions (IRa and IRb) of 25,476 bp separated by the single copy regions. The genome encodes 114 genes, 16 of which are duplicated. Most of the 85 protein-coding genes (CDS) had standard ATG start codons, while 3 genes including rps12, psbL and ndhD had abnormal start codons (ACT and ACG). In addition, a strong A/T bias was found in the majority of simple sequence repeats (SSRs) detected in the CP genome. Analysis of the phylogenetic relationships among 16 species revealed that L. chinense is a sister taxon to Lycium barbarum. Overall, the complete sequence and annotation of the L. chinense CP genome provides valuable genetic information to facilitate precise understanding of the taxonomy, species and phylogenetic evolution of the Solanaceae family.

Identification and Phylogenetic Analysis of the Complete Chloroplast Genomes of Three Ephedra Herbs Containing Ephedrine.

Ephedrae Herba and Ephedrae Radix et Rhizoma (Mahuang) have been used as Chinese herbal medicines. Ephedra plants mainly live in deserts and have good governance of desertification. Despite their important medicinal and environmental protection value, dietary supplements containing ephedrine from Ephedra species may threaten the health of people. Morphological resemblance amongst species causes difficulty in identifying the original species of Ephedra herbs. Chloroplast (CP) genome shows good prospects in identification and phylogenetic analysis. This study introduced the structures of the CP genomes of three Ephedra species and analysed their phylogenetic relationships. Three complete CP genomes of Ephedra showed four-part annular structures, namely, two single-copy regions and two inverted repeat regions. The entire CP genomes of three Ephedra species in terms of size were 109,550 bp (E. sinica), 109,667 bp (E. intermedia), and 109,558 bp (E. equisetina). Each CP genome of the three Ephedra species encoded 118 genes, including 73 protein-coding genes, 37 tRNA genes and 8 ribosomal RNA genes. Eleven high-variation regions were screened through mVISTA to be potential specific DNA barcodes for identifying Ephedra species. Maximum likelihood and maximum parsimony trees showed that CP genomes could be used to identify Ephedra species. The Ephedra species had a close phylogenetic relationship with Gnetum species and Welwitschia mirabilis. This research provided valuable information for the identification and phylogenetic analysis of gymnosperms and drug safety of Ephedra.

Development of Specific SNP Molecular Marker from Thistle in the DNA Sequences of Chloroplast TrnL-F and Matk Region Using HRM Analysis

Development of Specific SNP Molecular Marker from Thistle in the DNA Sequences of Chloroplast TrnL-F and Matk Region Using HRM Analysis