Identification of Polycistronic Transcriptional Units and Non-canonical Introns in Green Algal Chloroplasts Based on Long-read RNA Sequencing Data

2020 ◽  
Author(s):  
Xiaoxiao Zou ◽  
Heroen Verbruggen ◽  
Tianjingwei Li ◽  
Jun Zhu ◽  
Zuo Chen ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Green Algae ◽  
Higher Plants ◽  
Green Algal ◽  
Chloroplast Genomes ◽  
Caulerpa Lentillifera ◽  
Transcriptional Units ◽  
Group Ii ◽  
Distinct Features ◽  
Algal Chloroplast

Abstract Background: Chloroplasts are important semi-autonomous organelles in plants and algae. Unlike higher plants, the chloroplast genomes of green algal linage have distinct features both in organization and expression. Despite the architecture of chloroplast genome have been extensively studied in higher plants and several model species of algae, little is known about transcriptional features in green algal lineages. Results: Based on full-length cDNA (Iso-Seq) sequencing, we identified widely co-transcribed polycistronic transcriptional units (PTUs) in the green alga Caulerpa lentillifera. In addition to clusters of genes from the same pathway, we identified a series of PTUs of up to nine genes whose function in the plastid is not understood. The RNA data further allowed us to confirm widespread expression of fragmented genes and conserved open reading frames, which are both important features in green algal chloroplast genomes. In addition, a newly fragmented gene specific to C. lentillifera was discovered, which may represent a recent gene fragmentation event in chloroplast genome.Taking the accurate exon-intron boundary information, gene structural annotation was greatly improved across the siphonous green algae lineages. Our data also revealed a type of non-canonical Group II introns, with a deviant secondary structure and intronic ORFs lacking known splicing or mobility domains. These widespread introns have conserved positions in their genes and are excised precisely despite lacking clear consensus intron boundaries.Conclusion: Our study fills important knowledge gaps in chloroplast genome organization and transcription in green algae, and providing new insights into expression of polycistronic transcripts, freestanding ORFs and fragmented genes in algal chloroplast genomes. Moreover, we revealed an unusual type of Group II intron with distinct features and conserved positions in Bryopsidales. Our data represents interesting additions to knowledge of chloroplast intron structure and highlights clusters of uncharacterized genes that probably play important roles in plastid.

scholarly journals Identification of polycistronic transcriptional units and non-canonical introns in green algal chloroplasts based on long-read RNA sequencing data

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiaoxiao Zou ◽  
Heroen Verbruggen ◽  
Tianjingwei Li ◽  
Jun Zhu ◽  
Zou Chen ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Green Algae ◽  
Higher Plants ◽  
Green Algal ◽  
Chloroplast Genomes ◽  
Caulerpa Lentillifera ◽  
Transcriptional Units ◽  
Group Ii ◽  
Distinct Features ◽  
Algal Chloroplast

Abstract Background Chloroplasts are important semi-autonomous organelles in plants and algae. Unlike higher plants, the chloroplast genomes of green algal linage have distinct features both in organization and expression. Despite the architecture of chloroplast genome having been extensively studied in higher plants and several model species of algae, little is known about the transcriptional features of green algal chloroplast-encoded genes. Results Based on full-length cDNA (Iso-Seq) sequencing, we identified widely co-transcribed polycistronic transcriptional units (PTUs) in the green alga Caulerpa lentillifera. In addition to clusters of genes from the same pathway, we identified a series of PTUs of up to nine genes whose function in the plastid is not understood. The RNA data further allowed us to confirm widespread expression of fragmented genes and conserved open reading frames, which are both important features in green algal chloroplast genomes. In addition, a newly fragmented gene specific to C. lentillifera was discovered, which may represent a recent gene fragmentation event in the chloroplast genome. With the newly annotated exon-intron boundary information, gene structural annotation was greatly improved across the siphonous green algae lineages. Our data also revealed a type of non-canonical Group II introns, with a deviant secondary structure and intronic ORFs lacking known splicing or mobility domains. These widespread introns have conserved positions in their genes and are excised precisely despite lacking clear consensus intron boundaries. Conclusion Our study fills important knowledge gaps in chloroplast genome organization and transcription in green algae, and provides new insights into expression of polycistronic transcripts, freestanding ORFs and fragmented genes in algal chloroplast genomes. Moreover, we revealed an unusual type of Group II intron with distinct features and conserved positions in Bryopsidales. Our data represents interesting additions to knowledge of chloroplast intron structure and highlights clusters of uncharacterized genes that probably play important roles in plastids.

Recombinant Protein Production in Microalgae: Emerging Trends

2020 ◽  
Vol 27 (2) ◽  
pp. 105-110 ◽  
Author(s):  
Niaz Ahmad ◽  
Muhammad Aamer Mehmood ◽  
Sana Malik
Keyword(s):  
Chloroplast Genome ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Higher Plants ◽  
Scale Up ◽  
Chloroplast Transformation ◽  
Point Of View ◽  
Nuclear Transformation ◽  
Scale Production ◽  
Algal Chloroplast

: In recent years, microalgae have emerged as an alternative platform for large-scale production of recombinant proteins for different commercial applications. As a production platform, it has several advantages, including rapid growth, easily scale up and ability to grow with or without the external carbon source. Genetic transformation of several species has been established. Of these, Chlamydomonas reinhardtii has become significantly attractive for its potential to express foreign proteins inexpensively. All its three genomes – nuclear, mitochondrial and chloroplastic – have been sequenced. As a result, a wealth of information about its genetic machinery, protein expression mechanism (transcription, translation and post-translational modifications) is available. Over the years, various molecular tools have been developed for the manipulation of all these genomes. Various studies show that the transformation of the chloroplast genome has several advantages over nuclear transformation from the biopharming point of view. According to a recent survey, over 100 recombinant proteins have been expressed in algal chloroplasts. However, the expression levels achieved in the algal chloroplast genome are generally lower compared to the chloroplasts of higher plants. Work is therefore needed to make the algal chloroplast transformation commercially competitive. In this review, we discuss some examples from the algal research, which could play their role in making algal chloroplast commercially successful.

scholarly journals Purification and characterization of high- and low-molecular-mass isoforms of phosphoenolpyruvate carboxylase from Chlamydomonas reinhardtii

1998 ◽  
Vol 331 (1) ◽  
pp. 201-209 ◽  
Author(s):  
Jean RIVOAL ◽  
William C. PLAXTON ◽  
David H. TURPIN
Keyword(s):  
Green Algae ◽  
Phosphoenolpyruvate Carboxylase ◽  
Higher Plants ◽  
Current Model ◽  
Catalytic Subunit ◽  
Dihydroxyacetone Phosphate ◽  
Evolutionary Divergence ◽  
Banana Fruit ◽  
Higher Plant ◽  
Green Algal

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the supply of carbon skeletons for the assimilation of nitrogen by green algae. Two PEPC isoforms with respective native molecular masses of 400 (PEPC1) and 650 (PEPC2) kDa have been purified from Chlamydomonas reinhardtiiCW-15 cc1883 (Chlorophyceae). SDS/PAGE, immunoblot and CNBr peptide-mapping analyses indicate the presence of the same 100 kDa PEPC catalytic subunit in both isoforms. PEPC1 is a homotetramer, whereas PEPC2 seems to be a complex between the PEPC catalytic subunit and other immunologically unrelated polypeptides of 50–70 kDa. Kinetic analyses indicate that these PEPC isoforms are (1) differentially regulated by pH, (2) activated by glutamine and dihydroxyacetone phosphate and (3) inhibited by glutamate, aspartate, 2-oxoglutarate and malate. These results are consistent with the current model for the regulation of anaplerotic carbon fixation in green algae, and demonstrate that green algal PEPCs are uniquely regulated by glutamine. Several techniques were used to assess the structural relationships between C. reinhardtiiPEPC and the higher plant or prokaryotic enzyme. Immunoblot studies using anti-(green algal or higher plant PEPC) IgGs suggested that green algal (C. reinhardtii, Selenastrum minutum), higher plant (maize, banana fruit, tobacco) and prokaryotic (Synechococcus leopoliensis, Escherichia coli)PEPCs have little or no immunological relatedness. Moreover, the N-terminal amino acid sequence of the C. reinhardtiiPEPC subunit did not have significant similarity to the highly conserved corresponding region in enzymes from higher plants, and CNBr cleavage patterns of green algal PEPCs were distinct from those of higher plant and cyanobacterial PEPCs. These results point to significant evolutionary divergence between green algal, higher plant and prokaryotic PEPCs.

A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms

Genome ◽  
1999 ◽  
Vol 42 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Kurt Weising ◽  
Richard C Gardner
Keyword(s):  
Chloroplast Genome ◽  
Dna Sequences ◽  
Higher Plants ◽  
Pcr Primers ◽  
Universal Primers ◽  
Mononucleotide Repeat ◽  
Consensus Primer ◽  
Chloroplast Genomes ◽  
Pcr Products ◽  
Angiosperm Species

Short runs of mononucleotide repeats are present in chloroplast genomes of higher plants. In soybean, rice, and pine, PCR (polymerase chain reaction) with flanking primers has shown that the numbers of A or T residues in such repeats are variable among closely related taxa. Here we describe a set of primers for studying mononucleotide repeat variation in chloroplast DNA of angiosperms where database information is limited. A total of 39 (A)n and (T)n repeats (n [Formula: see text] 10) were identified in the tobacco chloroplast genome, and DNA sequences encompassing these 39 regions were aligned with orthologous DNA sequences in the databases. Consensus primer pairs were constructed and used to amplify total genomic DNA from a hierarchical set of angiosperms. All 10 primer pairs generated PCR products from members of the Solanaceae, and 8 of the 10 were also functional in most other angiosperm species. Levels of interspecific polymorphism within the genera Nicotiana, Lycopersicon (both Solanaceae), and Actinidia (Actinidiaceae) proved to be high, while intraspecific variation in Nicotiana tabacum, Lycopersicon esculentum, and Actinidia chinensis was limited. Sequence analysis of PCR products from three primer pairs revealed variable numbers of A, G, and T residues in mononucleotide arrays as the major cause of polymorphism in Actinidia. Our results suggest that universal primers targeted to mononucleotide repeats may serve as general tools to study chloroplast variation in angiosperms.Key words: genetic markers, chloroplast genome, microsatellites, consensus primers, angiosperms.

scholarly journals Structural variation and evolution of chloroplast tRNAs in green algae

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11524
Author(s):  
Fangbing Qi ◽  
Yajing Zhao ◽  
Ningbo Zhao ◽  
Kai Wang ◽  
Zhonghu Li ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Green Algae ◽  
Sequence Alignment ◽  
Structural Variation ◽  
Evolutionary Relationship ◽  
Multiple Sequence ◽  
Evolutionary Patterns ◽  
Systematic Analysis ◽  
Origin And Evolution ◽  
Green Algal

As one of the important groups of the core Chlorophyta (Green algae), Chlorophyceae plays an important role in the evolution of plants. As a carrier of amino acids, tRNA plays an indispensable role in life activities. However, the structural variation of chloroplast tRNA and its evolutionary characteristics in Chlorophyta species have not been well studied. In this study, we analyzed the chloroplast genome tRNAs of 14 species in five categories in the green algae. We found that the number of chloroplasts tRNAs of Chlorophyceae is maintained between 28–32, and the length of the gene sequence ranges from 71 nt to 91 nt. There are 23–27 anticodon types of tRNAs, and some tRNAs have missing anticodons that are compensated for by other types of anticodons of that tRNA. In addition, three tRNAs were found to contain introns in the anti-codon loop of the tRNA, but the analysis scored poorly and it is presumed that these introns are not functional. After multiple sequence alignment, the Ψ-loop is the most conserved structural unit in the tRNA secondary structure, containing mostly U-U-C-x-A-x-U conserved sequences. The number of transitions in tRNA is higher than the number of transversions. In the replication loss analysis, it was found that green algal chloroplast tRNAs may have undergone substantial gene loss during the course of evolution. Based on the constructed phylogenetic tree, mutations were found to accompany the evolution of the Green algae chloroplast tRNA. Moreover, chloroplast tRNAs of Chlorophyceae are consistent with those of monocotyledons and gymnosperms in terms of evolutionary patterns, sharing a common multi-phylogenetic pattern and rooted in a rich common ancestor. Sequence alignment and systematic analysis of tRNA in chloroplast genome of Chlorophyceae, clarified the characteristics and rules of tRNA changes, which will promote the evolutionary relationship of tRNA and the origin and evolution of chloroplast.

scholarly journals The plastid genome in Cladophorales green algae is encoded by hairpin plasmids

2017 ◽  
Author(s):  
Andrea Del Cortona ◽  
Frederik Leliaert ◽  
Kenny A. Bogaert ◽  
Monique Turmel ◽  
Christian Boedeker ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Green Algae ◽  
Plastid Genome ◽  
High Throughput Sequencing ◽  
Gc Content ◽  
Genome Architecture ◽  
Dna Molecules ◽  
Chloroplast Genes ◽  
Dna And Rna ◽  
Chloroplast Genomes

AbstractVirtually all plastid (chloroplast) genomes are circular double-stranded DNA molecules, typically between 100-200 kb in size and encoding circa 80-250 genes. Exceptions to this universal plastid genome architecture are very few and include the dinoflagellates where genes are located on DNA minicircles. Here we report on the highly deviant chloroplast genome of Cladophorales green algae, which is entirely fragmented into hairpin plasmids. Short and long read high-throughput sequencing of DNA and RNA demonstrated that the chloroplast genes of Boodlea composita are encoded on 1-7 kb DNA contigs with an exceptionally high GC-content, each containing a long inverted repeat with one or two protein-coding genes and conserved non-coding regions putatively involved in replication and/or expression. We propose that these contigs correspond to linear single-stranded DNA molecules that fold onto themselves to form hairpin plasmids. The Boodlea chloroplast genes are highly divergent from their corresponding orthologs. The origin of this highly deviant chloroplast genome likely occurred before the emergence of the Cladophorales, and coincided with an elevated transfer of chloroplast genes to the nucleus. A chloroplast genome that is composed only of linear DNA molecules is unprecedented among eukaryotes and highlights unexpected variation in the plastid genome architecture.

scholarly journals Chloroplast genome expansion by intron multiplication in the basal psychrophilic euglenoidEutreptiella pomquetensis

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3725 ◽  
Author(s):  
Nadja Dabbagh ◽  
Matthew S. Bennett ◽  
Richard E. Triemer ◽  
Angelika Preisfeld
Keyword(s):  
Chloroplast Genome ◽  
Green Algae ◽  
Gene Clusters ◽  
Single Copy ◽  
Protein Coding ◽  
Group Iii ◽  
Chloroplast Genomes ◽  
Plant Chloroplast ◽  
Intron Content ◽  
High Level

BackgroundOver the last few years multiple studies have been published showing a great diversity in size of chloroplast genomes (cpGenomes), and in the arrangement of gene clusters, in the Euglenales. However, while these genomes provided important insights into the evolution of cpGenomes across the Euglenales and within their genera, only two genomes were analyzed in regard to genomic variability between and within Euglenales and Eutreptiales. To better understand the dynamics of chloroplast genome evolution in early evolving Eutreptiales, this study focused on the cpGenome ofEutreptiella pomquetensis, and the spread and peculiarities of introns.MethodsTheEtl. pomquetensiscpGenome was sequenced, annotated and afterwards examined in structure, size, gene order and intron content. These features were compared with other euglenoid cpGenomes as well as those of prasinophyte green algae, includingPyramimonas parkeae.Results and DiscussionWith about 130,561 bp the chloroplast genome ofEtl. pomquetensis, a basal taxon in the phototrophic euglenoids, was considerably larger than the two other Eutreptiales cpGenomes sequenced so far. Although the detected quadripartite structure resembled most green algae and plant chloroplast genomes, the gene content of the single copy regions inEtl. pomquetensiswas completely different from those observed in green algae and plants. The gene composition ofEtl. pomquetensiswas extensively changed and turned out to be almost identical to other Eutreptiales and Euglenales, and not toP. parkeae. Furthermore, the cpGenome ofEtl. pomquetensiswas unexpectedly permeated by a high number of introns, which led to a substantially larger genome. The 51 identified introns ofEtl. pomquetensisshowed two major unique features: (i) more than half of the introns displayed a high level of pairwise identities; (ii) no group III introns could be identified in the protein coding genes. These findings support the hypothesis that group III introns are degenerated group II introns and evolved later.

scholarly journals Do Red and Green Make Brown?: Perspectives on Plastid Acquisitions within Chromalveolates

2011 ◽  
Vol 10 (7) ◽  
pp. 856-868 ◽  
Author(s):  
Richard G. Dorrell ◽  
Alison G. Smith
Keyword(s):  
Net Primary Productivity ◽  
Algal Species ◽  
Biodiesel Production ◽  
Selective Pressures ◽  
Green Algal ◽  
Chloroplast Genomes ◽  
Large Numbers ◽  
Red Algal ◽  
Algal Endosymbiont ◽  
Algal Chloroplast

ABSTRACT The chromalveolate “supergroup” is of key interest in contemporary phycology, as it contains the overwhelming majority of extant algal species, including several phyla of key importance to oceanic net primary productivity such as diatoms, kelps, and dinoflagellates. There is also intense current interest in the exploitation of these algae for industrial purposes, such as biodiesel production. However, the evolution of the constituent species, and in particular the origin and radiation of the chloroplast genomes, remains poorly understood. In this review, we discuss current theories of the origins of the extant red alga-derived chloroplast lineages in the chromalveolates and the potential ramifications of the recent discovery of large numbers of green algal genes in chromalveolate genomes. We consider that the best explanation for this is that chromalveolates historically possessed a cryptic green algal endosymbiont that was subsequently replaced by a red algal chloroplast. We consider how changing selective pressures acting on ancient chromalveolate lineages may have selectively favored the serial endosymbioses of green and red algae and whether a complex endosymbiotic history facilitated the rise of chromalveolates to their current position of ecological prominence.

scholarly journals The inflated mitochondrial genomes of siphonous green algae reflect processes driving expansion of noncoding DNA and proliferation of introns

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8273 ◽  
Author(s):  
Sonja I. Repetti ◽  
Christopher J. Jackson ◽  
Louise M. Judd ◽  
Ryan R. Wick ◽  
Kathryn E. Holt ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Gene Arrangement ◽  
Mitochondrial Genomes ◽  
Effective Population ◽  
Noncoding Dna ◽  
Bioinformatic Tools ◽  
Evolutionary Forces ◽  
Green Algal ◽  
Chloroplast Genomes ◽  
Caulerpa Lentillifera

Within the siphonous green algal order Bryopsidales, the size and gene arrangement of chloroplast genomes has been examined extensively, while mitochondrial genomes have been mostly overlooked. The recently published mitochondrial genome of Caulerpa lentillifera is large with expanded noncoding DNA, but it remains unclear if this is characteristic of the entire order. Our study aims to evaluate the evolutionary forces shaping organelle genome dynamics in the Bryopsidales based on the C. lentillifera and Ostreobium quekettii mitochondrial genomes. In this study, the mitochondrial genome of O. quekettii was characterised using a combination of long and short read sequencing, and bioinformatic tools for annotation and sequence analyses. We compared the mitochondrial and chloroplast genomes of O. quekettii and C. lentillifera to examine hypotheses related to genome evolution. The O. quekettii mitochondrial genome is the largest green algal mitochondrial genome sequenced (241,739 bp), considerably larger than its chloroplast genome. As with the mtDNA of C. lentillifera, most of this excess size is from the expansion of intergenic DNA and proliferation of introns. Inflated mitochondrial genomes in the Bryopsidales suggest effective population size, recombination and/or mutation rate, influenced by nuclear-encoded proteins, differ between the genomes of mitochondria and chloroplasts, reducing the strength of selection to influence evolution of their mitochondrial genomes.

scholarly journals Screening and verification of extranuclear genetic markers in green tide algae from the Yellow Sea

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0250968
Author(s):  
Chuner Cai ◽  
Kai Gu ◽  
Hui Zhao ◽  
Sophie Steinhagen ◽  
Peimin He ◽  
...  
Keyword(s):  
Phylogenetic Tree ◽  
Chloroplast Genome ◽  
Genetic Markers ◽  
Yellow Sea ◽  
Algal Species ◽  
The Yellow Sea ◽  
Evolutionary Trends ◽  
Green Tides ◽  
Green Algal ◽  
Chloroplast Genomes

Over the past decade, Ulva compressa, a cosmopolitan green algal species, has been identified as a component of green tides in the Yellow Sea, China. In the present study, we sequenced and annotated the complete chloroplast genome of U. compressa (alpha-numeric code: RD9023) and focused on the assessment of genome length, homology, gene order and direction, intron size, selection strength, and substitution rate. We compared the chloroplast genome with the mitogenome. The generated phylogenetic tree was analyzed based on single and aligned genes in the chloroplast genome of Ulva compared to mitogenome genes to detect evolutionary trends. U. compressa and U. mutabilis chloroplast genomes had similar gene queues, with individual genes exhibiting high homology levels. Chloroplast genomes were clustered together in the entire phylogenetic tree and shared several forward/palindromic/tandem repetitions, similar to those in U. prolifera and U. linza. However, U. fasciata and U. ohnoi were more divergent, especially in sharing complementary/palindromic repetitions. In addition, phylogenetic analyses of the aligned genes from their chloroplast genomes and mitogenomes confirmed the evolutionary trends of the extranuclear genomes. From phylogenetic analysis, we identified the petA chloroplast genes as potential genetic markers that are similar to the tufA marker. Complementary/forward/palindromic interval repetitions were more abundant in chloroplast genomes than in mitogenomes. Interestingly, a few tandem repetitions were significant for some Ulva subspecies and relatively more evident in mitochondria than in chloroplasts. Finally, the tandem repetition [GAAATATATAATAATA × 3, abbreviated as TRg)] was identified in the mitogenome of U. compressa and the conspecific strain U. mutabilis but not in other algal species of the Yellow Sea. Owing to the high morphological plasticity of U. compressa, the findings of this study have implications for the rapid non-sequencing detection of this species during the occurrence of green tides in the region.

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