Phylogenetic study on Scenedesmacae with the description of a new genus Coccoidesmus gen.nov.(Chlorophyceae,Chlorophyta)and chloroplast genome analyses

Members of the family Scenedesmaceae are some of the most common algal taxa in inland ecosystems,and they are widely distributed in freshwaters,aerial,and sub-aerial habitats.With the continuous updating of methods,the classic morphological taxonomy of this family needs to be revised.In recent years,many genera of Scenedesmaceae have been established via the use of molecular methods.The phylogenetic relationships within Scenedesmaceae were analyzed using different molecular markers and morphological data,and the new freshwater genus Coccoidesmus Wang,Hou et Liu gen.nov.was described.Two new species in this genus were also described.Phylogenetic analysis based on tufA genes revealed that the new genus formed an independent clade closely related to Comasiella.However,these two genera are characterized by significant morphological differences in colony arrangement and cell shape.The chloroplast genome of the type species was assembled and annotated,and analyses of genome structure and sequences were conducted.More genome data could help clarify the phylogenetic relationships within this family.

A Golden2-like transcription factor, BnGLK1a, improves chloroplast development, photosynthesis, and seed weight in rapeseed

Enhancing photosynthetic efficiency is a major goal for improving crop yields under agricultural field conditions and is associated with chloroplast biosynthesis and development.In this study,we demonstrate that Golden2-like 1a(BnGLK1a)plays an important role in regulating chloroplast development and photosynthetic efficiency.Overexpressing BnGLK1a resulted in significant increases in chlorophyll content,the number of thylakoid membrane layers and photosynthetic efficiency in Brassica napus,while knocking down BnGLK1a transcript levels through RNA interference(RNAi)had the opposite effects.A yeast two-hybrid screen revealed that BnGLK1a interacts with the abscisic acid receptor PYRABACTIN RESISTANCE 1-LIKE 1-2(BnPYL1-2)and CONSTITUTIVE PHOTOMORPHOGENIC 9 SIGNALOSOME 5A subunit(BnCSN5A),which play essential roles in regulating chloroplast development and photosynthesis.Consistent with this,BnGLK1a-RNAi lines of B.napus display hypersensitivity to the abscisic acid(ABA)response.Importantly,overexpression of BnGLK1a resulted in a 10%increase in thousand-seed weight,whereas seeds from BnGLK1a-RNAi lines were 16%lighter than wild type.We propose that BnGLK1a could be a potential target in breeding for improving rapeseed productivity.Our results not only provide insights into the mechanisms of BnGLK1a function,but also offer a potential approach for improving the productivity of Brassica species.

Comparative and Phylogenetic Analysis of the Complete Chloroplast Genomes of 19 Species in Rosaceae Family

Rosaceae represents a vast and complex group of species,with its classification being intricate and contentious.The taxonomic placement of many species within this family has been a subject of ongoing debate.The study utilized the Illumina platform to sequence 19 plant species from 10 genera in the Rosaceae.The cp genomes,vary-ing in size from 153,366 to 159,895 bp,followed the typical quadripartite organization consisting of a large single-copy(LSC)region(84,545 to 87,883 bp),a small single-copy(SSC)region(18,174 to 19,259 bp),and a pair of inverted repeat(IR)regions(25,310 to 26,396 bp).These genomes contained 132–138 annotated genes,including 87 to 93 protein-coding genes(PCGs),37 tRNA genes,and 8 rRNA genes using MISA software,52 to 121 simple sequence repeat(SSR)loci were identified.D.arbuscular contained the least of SSRs and did not have hexanotides,A.lineata contained the richest SSRs.Long terminal repeats(LTRs)were primarily composed of palindromic and forward repeat sequences,meanwhile,The richest LTRs were found in Argentina lineata.Except for Argentina lineata,Fragariastrum eriocarpum,and Prunus trichostoma,which varied in gene type and position on both sides of the boundary,the remaining species were found to be mostly conserved according to IR boundary analysis.The examination of the Ka/Ks ratio revealed that only the infA gene had a value greater than 1,indicating that this gene was primarily subjected to positive selection during evolution.Additionally,9 hotspots of variation were identified in the LSC and SSC regions.Phylogenetic analysis confirmed the scientific validity of the genus Prunus L.sensu lato(s.l.)within the Rosaceae family.The separation of the three genera Argentina Hill,Fragariastrum Heist.ex Fabr.and Dasiphora Raf.from Potentilla L.may be a more scientific classification.These results offer fresh perspectives on the taxonomy of the Rosaceae.

Structural Characterization of Chloroplast Genome in Alpinia japonica(Thunb.)Miq.,a Medicinal Plant of the Genus Alpinia

The analysis of chloroplast gene characteristics in Alpinia japonica(Thunb.)Miq.is of great significance for developing relevant genetic resources.The high-throughput sequencing and bioinformatic research were performed to analyze the chloroplast genome characteristics of A.japonica.The total chloroplast genome length of A.japonica was 161,906 bp,with a typical circular tetrameric structure.And 133 genes were annotated,comprising 87 protein-coding,38 tRNA,and 8 rRNA genes.Furthermore,22 genes contained two copies,and 18 genes owned introns.Repeat sequence analysis showed that it contains 321 simple sequence repeats(SSRs)and 37 long segment repeats.Compared with the chloroplast genomes of eight representative plants in the genus Alpinia,the gene structure,type,and quantity were relatively conservative.Rps12 was the highest variation site in the entire chloroplast gene.A phylogenetic tree showed that the genus Alpinia was the most closely related to the genus Amomum.Meanwhile,A.japonica is the most closely related to Alpinia chinensis belonging to the genus Alpinia.Overall,the chloroplast genome of a new species was reported in the genus Alpinia,and a basis was provided for the utilization of Alpinia plants as a medical resource.

Os DXR interacts with Os MORF1 to regulate chloroplast development and the RNA editing of chloroplast genes in rice

Plant chlorophyll biosynthesis and chloroplast development are two complex processes that are regulated by exogenous and endogenous factors. In this study, we identified OsDXR, a gene encoding a reductoisomerase that positively regulates chlorophyll biosynthesis and chloroplast development in rice. OsDXR knock-out lines displayed the albino phenotype and could not complete the whole life cycle process. OsDXR was highly expressed in rice leaves, and subcellular localization indicated that OsDXR is a chloroplast protein. Many genes involved in chlorophyll biosynthesis and chloroplast development were differentially expressed in the OsDXR knock-out lines compared to the wild type.Moreover, we found that the RNA editing efficiencies of ndhA-1019 and rpl2-1 were significantly reduced in the OsDXR knock-out lines. Furthermore, OsDXR interacted with the RNA editing factor OsMORF1 in a yeast two-hybrid screen and bimolecular fluorescence complementation assay. Finally, disruption of the plastidial 2-C-methyl-derythritol-4-phosphate pathway resulted in defects in chloroplast development and the RNA editing of chloroplast genes.

PtrDJ1C,an atypical member of the DJ-1 superfamily,is essential for early chloroplast development and lignin deposition in poplar

The nuclear-encoded factors and the photosynthetic apparatus have been studied extensively during chloroplast biogenesis.However,many questions regarding these processes remain unanswered,particularly in perennial woody plants.As a model material of woody plants,poplar not only has very significant value of research,but also possesses economic and ecological properties.This study reports the Populus trichocarpa DJ-1C(PtrDJ1C)factor,encoded by a nuclear gene,and a member of the DJ-1 superfamily.PtrDJ1C knock-out with the CRISPR/Cas9 system resulted in different albino phenotypes.Chlorophyll fluorescence and immunoblot analyses showed that the levels of photosynthetic complex proteins decreased significantly.Moreover,the transcript level of plastid-encoded RNA polymerase-dependent genes and the splicing efficiency of several introns were affected in the mutant line.Furthermore,rRNA accumulation was abnormal,leading to developmental defects in chloroplasts and affecting lignin accumulation.We concluded that the PtrDJ1C protein is essential for early chloroplast development and lignin deposition in poplar.

OsPPR9 encodes a DYW-type PPR protein that affects editing efficiency of multiple RNA editing sites and is essential for chloroplast development

Photosynthesis occurs mainly in chloroplasts,whose development is regulated by proteins encoded by nuclear genes.Among them,pentapeptide repeat(PPR)proteins participate in organelle RNA editing.Although there are more than 450 members of the PPR protein family in rice,only a few affect RNA editing in rice chloroplasts.Gene editing technology has created new rice germplasm and mutants,which could be used for rice breeding and gene function study.This study evaluated the functions of OsPPR9 in chloroplast RNA editing in rice.The osppr9 mutants were obtained by CRISPR/Cas9,which showed yellowing leaves and a lethal phenotype,with suppressed expression of genes associated with chloroplast development and accumulation of photosynthetic-related proteins.In addition,loss of OsPPR9 protein function reduces the editing efficiency of rps8-C182,rpoC2-C4106,rps14-C80,and ndhB-C611 RNA editing sites,which affects chloroplast growth and development in rice.Our data showed that OsPPR9 is highly expressed in rice leaves and encodes a DYW-PPR protein localized in chloroplasts.Besides,the OsPPR9 protein was shown to interact with OsMORF2 and OsMORF9.Together,our findings provide insights into the role of the PPR protein in regulating chloroplast development in rice.

Advancing approach and toolbox in optimization of chloroplast genetic transformation technology

Chloroplast is a discrete,highly structured,and semi-autonomous cellular organelle.The small genome of chloroplast makes it an up-and-coming platform for synthetic biology.As a special means of synthetic biology,chloroplast genetic engineering shows excellent potential in reconstructing various sophisticated metabolic pathways within the plants for specific purposes,such as improving crop photosynthetic capacity,enhancing plant stress resistance,and synthesizing new drugs and vaccines.However,many plant species exhibit limited efficiency or inability in chloroplast genetic transformation.Hence,new transformation technologies and tools are being constantly developed.In order to further expand and facilitate the application of chloroplast genetic engineering,this review summarizes the new technologies in chloroplast genetic transformation in recent years and discusses the choice of appropriate synthetic biological elements for the construction of efficient chloroplast transformation vectors.

OsTHA8 encodes a pentatricopeptide repeat protein required for RNA editing and splicing during rice chloroplast development

In higher plants, the chloroplast is the most important organelle for photosynthesis and for numerous essential metabolic processes in the cell. Although many genes involved in chloroplast development have been identified, the mechanisms underlying such development are not fully understood. In this study, a rice(Oryza sativa) mutant exhibiting pale green color and seedling lethality was isolated from a mutant library. The mutated gene was identified as an ortholog of THA8(thylakoid assembly 8) in Arabidopsis and maize. This gene is designated as OsTHA8 hereafter. OsTHA8 showed a typical pentatricopeptide repeat(PPR) characteristic of only four PPR motifs. Inactivation of OsTHA8 led to a deficiency in chloroplast development in the rice seedling stage. OsTHA8 was expressed mainly in young leaves and leaf sheaths.The OsTHA8 protein was localized to the chloroplast. Loss of function of OsTHA8 weakened the editing efficiency of ndhB-611/737 and rps8-182 transcripts under normal conditions. Y2H and BiFC indicated that OsTHA8 facilitates RNA editing by forming an editosome with multiple organellar RNA editing factor(OsMORF8) and thioredoxin z(OsTRXz), which function in RNA editing in rice chloroplasts. Defective OsTHA8 impaired chloroplast ribosome assembly and resulted in reduced expression of PEP-dependent genes and photosynthesis-related genes. Abnormal splicing of the chloroplast gene ycf3 was detected in ostha8. These findings reveal a synergistic regulatory mechanism of chloroplast biogenesis mediated by RNA, broaden the function of the PPR family, and shed light on the RNA editing complex in rice.

Research Progress of Sugarcane Chloroplast Genome

Along with the development of modern molecular biology technologies, complete chloroplast genomes have been sequenced in various plant species to date, and the structure, function and expression of these genes have been deter-mined. The chloroplast genome structure in most higher plants is stable, since the gene number, arrangement and composition are conservative. The determination of sugarcane chloroplast genome sequence laid a good foundation for sugarcane chloroplast related research. This article gives a review on the research progress of sugarcane chloroplast genome through the chloroplast genome map, gene structure, function, chloroplast RNA editing, and phylogenetic analysis in Saccharum and relat-ed genera. This study held great potential to clarify more directions in researches, including sugarcane chloroplast genetic transformation, complete chloroplast nu-cleotide sequence determination in Saccharum and closely related genera, cpSSRs development and application.

cpSSR: a New Tool to Analyze Chloroplast Genome of Citrus Somatic Hybrids

Chloroplast simple sequence repeat (cpSSR) markers in Citrus were developed and successfully used to analyze chloroplast genome inheritance of Citrus somatic hybrids. Twenty-two previously reported cpSSR primer pairs from pine (Pinus thunbergii Parl.), rice (Otyza sativa L.) and tobacco (Nicotiana tabacum L.) were tested in Citrus, nine of which could amplify intensive PCR products by agarose gel electrophoresis. Chloroplast genome inheritance of Citrus somatic hybrids from nine fusions was then analyzed, and five of the nine pre-screened primer pairs showed polymorphisms by polyacrylamide gel electrophoresis. The results revealed the random inheritance nature of chloroplast genome in all analyzed Citrus somatic hybrids, which was in agreement with previous reports based on RFLP or CAPS analyses. It was also shown that cpSSR is a more efficient tool in chloroplast genome analyses of somatic hybrids in higher plants, compared with the conventional RFLP or CAPS analyses.

Inheritance of Chloroplast and Mitochondrial DNA in Chinese Fir (Cunninghamia lanceolata)

The inheritance of mitochondrial (mt) DNA and chloroplast (cp) DNA was investigated in intergeneric hybrids from crossing between Cunninghamia lanceolata (Lamb.) Hook. and Cryptomeria fortunei Hooibrenk. The chloroplast trnL trnF region and one intra genic segment of the mitochondrial gene, Cox Ⅲ, were amplified from those of the parents and hybrids by PCR using gene specific primers. Cp and mtDNA polymorphisms of the amplified regions were detected between the parents after restriction digestions. Restriction fragment length polymorphism (RFLP) analysis revealed that all the F 1 individuals possessed Cox Ⅲ restriction fragment patterns (characteristic of the paternal parent Cryptomeria fortunei ) and the trnL trnF region (identical to the maternal parent Cunninghamia lanceolata ) showing that a different mode of inheritance for organelle DNA has occurred in the hybrids. Furthermore, the maternal inheritance of chloroplast DNA is reported here for the first time in coniferophyta.

Effects of High K^+ and Alkaline pH on Ultrastructure of Dunaliella salina Chloroplasts

ItwasreportedthatthegrowthofDunaliellasalinaTeod .culturedinmediumcontaining 1mol/L NaClwasalmostcompletelyinhibitedbytheadditionof 10 0mmol/LKCl.ThehighK+ (10 0mmol/LKCl) treatmentalsosignificantlyinhibitedthephotosyntheticrateofD .salinaanddecreasedchlorophyllcontentsin algae .ThisstudyfocusesonpossibleeffectsofhighK+ oralkalinepHontheultrastructuralchangeofchloro plastsinD .salina .AfterD .salinawasculturedinamediumcontaining 10 0mmol/LKClorinamedium withalkalinepHfor 8to 10days ,dramaticultrastructuralchangesoccurredinthechloroplastsincludingthy lakoidswelling ,volumeincreaseofchloroplast,andsignificantaccumulationofstarchgrainsinchloroplasts . Theresultsareconsistentwithourpreviousreportindicatingthattheultrastructuralchangesinchloroplastun derhighK+ oralkalinepHmayleadtoaninhibitoryeffectsonphotosynthesisandoverallgrowthofD .sali na .

Expression of the Thylakoid Membrane Localized PPF1 in Transgenic Arabidopsis Affects Chloroplast Development

PPF1 is a vegetative growth related gene that encodes a putative membrane protein having high homology with Arabidopsis chloroplast thylakoid protein ALB3. Immunoelectron microscopic assay showed that PPF1 was mainly localized in the thylakold membrane and was highly expressed in well-developed chloroplasts of short day (SD) grown G2 pea while having a very low abundance in chloroplasts of long day (LD) grown plants two weeks after flowering. Comparison of the leaf senescence processes in transgenic Arabidopsis and wild type plants revealed that overexpression of PPF1 delayed leaf senescence, while the depression of its Arabidopsts homologue (ALB3) with PPF1 antisense mRNA accelerated leaf senescence obviously. Ultrastructural analysis of transgenic Arabidopsis plants showed that when PPF1 was overexpressed in Arabidopsis, the chloroplasts were bigger and had much more grana and stroma thylakoid membranes than those of wild type plants. On the contrary, when PPF1 was expressed in antisense orientation to reduce the level of PPF1 homologue in Arabidopsis, the transgenic plants had smaller chloroplasts With less grana. and poorly developed thylakoid membrane systems. These results suggested that the developmental status of chloroplasts was positively correlated with the level of PPF1 or its Arabidopsts homologue, ALB3. Our results suggested that PPF1 gene might regulate plant development by controlling chloroplast development.

Changes of Chorophyll protein Complexes and Photosynthetic Activities of Chloroplasts from Lotus (Nelumbo nucifera) Seeds Germinating in Light

The changes of chlorophyll_protein complexes and photosynthetic activities of chloroplast isolated from lotus ( Nelumbo nucifera Gaertn.) seeds germinating under illumination were studied. SDS PAGE analysis of chlorophyll_protein complexes showed that there was only the light harvesting chlorophyll a/b protein complex from PSⅡ (LHCⅡ) precursor in chloroplast from lotus seeds germinated for 2 to 6 days, while LHC Ⅱ 1, and the chlorophyll_protein complex of PSⅠ (CPⅠ) appeared on the 8th day of germination and PSⅡ reaction center complex appeared later. Studies on the polypeptides composition of the chloroplast revealed the following results: 1) Small amount of the 27 kD polypeptide was synthesized in invisible light; 2) The 30 kD polypeptide existed previously in the plumules of the dry seeds; 3) The amount of the 30 kD polypeptide was more than any other polypeptides before germination and decreased gradually throughout germination, while the 27 kD polypeptide changed in the opposite way; 4) In the process of germination, measurement of the electron transport rate and the fluorescence induction kinetics at room temperature showed that PSⅡ activities and efficiency of primary light energy transformation were only experimentally measurable after 7 days of germination and gradually increased afterwards. At the same time, the chl a/b ratio rose from the lower value to normal; 5) The changes of chloroplast membrane components and its functions are concomitant in concert with that of the ultrastructure of chloroplast membranes during germination, as shown in our earlier work . The results have proved again that a different developmental pathway of chloroplast is likely to exist in the lotus plumules, which might provide an important clue for N. nucifera in having an unique position in the phylogeny of the angiosperm.

Genetic Diversity of Indocalamus Determined by Chloroplast DNA Sequence

[Objective]The aim was to research the relationship and genetic diversity of Indocalamus.[Method]Using 13 samples of Indocalamus and 3 samples of Sasa as materials,the intergenic regions of trnL-trnF gene in chloroplast were amplified by PCR,and sequence analysis and phylogenetic trees construction were carried out.[Result]Using the universal primer,the intergenic regions of trnL-trnF were amplified,the lengths of the segments varied from 1 008 bp to 1 103 bp,of which 940 bp was compared.The dendrogram of trnL-trnF sequences showed that Indocalamus and Sasa were clustered together and they were homologous by 99%.All the samples were divided into five groups,the first group included 12 samples such as Indosalamus pedalis,I.pumilus,I.victorialis,I.longiauritus,I.tessellatus,Sasa sinica,Sasa pygmaea,I.barbatus,I.guangdongensis,I.herklotsii,I.Hirtivaginatus and S.fortunei.I.decorus,I.lacunosus,I.Latifolius and I.Migoi were respectively divided into four groups.[Conclusion]The high homology of all samples showed the low evolution speed and little information sites which suggested that the phylogeny of Indocalamus could not be well resolved by the intergenic region of trnL-trnF.

Construction of Double Cross-over Expression Vector for Chloroplast Multicistron in Brassica napus L.

[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Method] Two primers were designed based on the known Brassica napus chloroplast DNA sequences AF267640 and Z50868 in GenBank. By using PCR method, two Brassica napus L. chloroplast DNA fragments were obtained, which were named RbcL and ACCD. The two Brassica na- pus chloroplast DNA homologous fragments were then cloned into plasmid pMD18-T to obtain recombinant plasmid pHBM715. Tandem expression cassette harboring spectinomycin-resistant gene aadA, mannanase gene man and green fluorescent pro- tein gene gfp was cloned into the plasmid pHBM715, thereby constructing Brassica napus chloroplast multicistron double cross-over expression vector pHBM716, which was transformed into Escherichia coil for expression and identification. [Result] Plate qualitative analysis was conducted for the functional identification of expression cas- sette in the constructed Brassica napus chloroplast multicistron double cross-over ex- pression vector, results showed that the three genes of the same multicistron were all expressed in E. coil [Conclusion] This study successfully constructed Brassica napus chloroplast multicistron double cross-over expression vector, which laid the foundation for the genetic engineering of Brassica napus chloroplast.

Effects of Doubled_CO_2 Concentration on Ultrastructure, Supramolecular Architecture and Spectral Characteristics of Chloroplasts from Wheat

Wheat ( Triticum aestivum L.) plants were grown under ambient and doubled_CO 2(plus 350 μL/L) concentration in cylindrical open_top chamber to examine their effects on the ultrastructure, supramolecular architecture, absorption spectrum and low temperature (77 K) fluorescence emission spectrum of the chloroplasts from wheat leaves. The results were briefly summarized as follows: (1) The wheat leaves possessed normally developed chloroplasts with intact grana and stroma thylakoid membranes; The grana intertwined with stroma thylakoid membranes and increased slightly in stacking degree and the width of granum, in spite of more accumulated starch grains within the chloroplasts than those in control; (2) The particle density in the stacked region of the endoplasmic fracture face (EFs) and protoplasmic fracture face (PFs) and in the unstacked region the endoplasmic fracture face (EFu) and the protoplasmic fracture face (PFu) was significantly higher than that of control. Furthermore, in some cases many more particles on EFs faces of thylakoid membranes appeared as a paracrystalline particle array; (3) The variations in the structure of chloroplasts were consistent with the absorption spectra and the low temperature (77 K) fluorescence emission spectra of the chloroplasts developed under the doubled_CO 2 concentration. Results indicate that the capability of light energy absorption of chloroplasts and regulative capability of excitation energy distribution between PSⅡ and PSⅠ were raised by doubled_CO 2 concentration. This is very favorable for final productivity of wheat.

MECHANISM OF CHANGE IN THE CATION INDUCED EXCITA TION ENERGY DISTRIBUTION BETWEEN PSⅡ AND PSⅠ IN THE CHLOROPLAST MEMBRANE FROM ZOSTERA MARINA *

The interrelations between thylakoid polypeptide components and Mg 2+ induced Chl a fluorescence and thylakoid surface charge changes were investigated in Zostera marina chloroplasts treated with Ca 2+ and trypsin. It was observed that:1. The increase of Mg 2+ induced PSⅡ fluorescence intensity was closely related to the decrease of Mg 2+ induced surface charge density of the thylakoid membrane in the normal chloroplast; 2. Removal of the 32~34 kD polypeptides of the thylakoid surface by Ca 2+ extraction of the chloroplast did not affect the Mg 2+ induced phenomena; 3. If the Ca 2+ treated chloroplast was further digested by trypsin to remove the 26 kD polypeptide of the membrane surface, the Mg 2+ induced phenomena disappeared completely. These results clearly indicated that the 26 kD polypeptide of thylakoid surface is the specific acting site of the cation that induced these two correlated phenomena in the chloroplast from Zostera marina. The mechanism on the regulating effect of the cation on excitation energy distribution between PSⅡ and PSⅠ was discussed.

Chloroplast Genetic Engineering in Higher Plants

Chloroplast genetic engineering, with several advantages over nuclear genetic engineering, is now regarded as an attractive new technology in basic and applied research, including deepening our understanding of plastid genome, engineering plant metabolic system, generating transplastomic plants with higher resistance to insect, disease, drought and herbicide and bioproducing of antibodies and vaccines. In this review, the principle and operating system for chloroplast genetic engineering and its application in higher plants have been discussed.