No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Identification and expression analysis of genome-wide long noncoding RNA responsive CO2 fluctuated environment in marine microalga Nannochloropsis oceanica
Abstract
Long non-coding RNAs (lncRNAs) have been demonstrated to participate in plant growth and development as well as response to different biotic and abiotic stresses. However, the knowledge of lncRNA was limited in microalgae. In this study, by RNA deep sequencing, 134 lncRNAs were identified in marine Nannochloropsis oceanica in response to carbon dioxide fluctuation. Among them, there were 51 lncRNAs displayed differentially expressed between low and high CO2 treatments, including 33 upregulation and 18 downregulation lncRNAs. Cellulose metabolic process, glucan metabolic process, polysaccharide metabolic process, and transmembrane transporter activity were functionally enriched. Multiple potential target genes of lncRNA and lncRNA-mRNA co-located gene network were analyzed. Subsequent analysis had demonstrated that lncRNAs would participate in many biological molecular processes, including gene expression, transcriptional regulation, protein expression and epigenetic regulation. In addition, alternative splicing events were firstly analyzed in response to CO2 fluctuation. There were 2051 alternative splicing (AS events) identified, which might be associated with lncRNA. These observations will provide a novel insight into lncRNA function in Nannochloropsis and provide a series of targets for lncRNA-based gene editing in future.
... LncRNAs involve in very diverse and important functions in many key biological processes, including epigenetic regulation, localization regulation, and protein function. LncR-NAs also act as regulators of gene expression in various aspects of plant development (Baruah et al. 2021;Dykes and Emanueli 2017;Wei et al. 2022). We, in the previous study, attempted to address the effects of UV-B radiation on the V. carteri whole transcriptome coding genes' profile (Ekhtari et al. 2019). ...
... In recent years, benefiting from the rapid development of high-throughput sequencing technology, RNA-seq, and computational analysis methods, more lncRNAs were identified and discovered in various plant species, including Zea mays, Populus tomentosa, and Actinidia chinensis (Ding et al. 2019;Jain, et al. 2021;Zhu et al. 2022). Identifying and analyzing lncRNAs under different conditions such as nutrient stresses, light intensity, and light quality have been investigated in Arabidopsis, wheat, rice, maize, cotton, Medicago, and peanut (Wei et al. 2022). In previous studies related to the identification (Lu et al. 2017), 2040 lncRNAs in Brassica rapa (Liu et al. 2018), 1323 novel lncRNAs in Ginkgo biloba leaves (Wang et al. 2018), 4648 lncRNAs in Physcomitrella patens (Simopoulos et al. 2019), 381 lncRNAs in Pteris vittate (Yan et al. 2019), and 1626 and 2208 putative high confidence lncRNAs in root and leaf of rice (Oryza sativa L.) (Jain, et al. 2021) were identified and reported. ...
... So far, few reports and studies related to lncRNAs in algae and microalgae are available. In previous studies, 1440 high-confidence lncRNAs in unicellular green alga Chlamydomonas reinhardtii (Li et al. 2016), 717 putative lncRNAs in brown alga Ectocarpus genome (Cormier et al. 2017), 4138 lncRNAs in marine diatom Phaeodactylum tricornutum (Cruz de Carvalho and Bowler 2020), and 134 novel lncRNAs in the marine microalga Nannochloropsis oceanica (Wei et al. 2022) have been identified. However, our knowledge regarding the lncRNAs in algae is limited. ...
Long non-coding RNAs (lncRNAs) are identified as important regulatory molecules related to diverse biological processes. In recent years, benefiting from the rapid development of high-throughput sequencing technology, RNA-seq, and analysis methods, more lncRNAs have been identified and discovered in various plant and algal species. However, so far, only limited studies related to algal lncRNAs are available. Volvox carteri f. nagariensis is the best multicellular model organism to study in developmental and evolutionary biology; therefore, studying and increasing information about this species is important. This study identified lncRNAs in the multicellular green algae Volvox carteri and 1457 lncRNAs were reported, using RNA-seq data and with the help of bioinformatics tools and software. This study investigated the effect of low-dose UV-B radiation on changes in the expression profile of lncRNAs in gonidial and somatic cells. The differential expression of lncRNAs was analyzed between the treatment (UV-B) and the control (WL) groups in gonidial and somatic cells. A total of 37 and 26 lncRNAs with significant differential expression in gonidial and somatic cells, respectively, were reported. Co-expression analysis between the lncRNAs and their neighbor protein-coding genes (in the interval of ± 10 Kb) was accomplished. In gonidial cells, 184 genes with a positive correlation and 13 genes with a negative correlation (greater than 0.95), and in somatic cells, 174 genes with a positive correlation, and 18 genes with a negative correlation were detected. Functional analysis of neighboring coding genes was also performed based on gene ontology. The results of the current work may help gain deeper insight into the regulation of gene expression in the studied model organism, Volvox carteri.
... Microalgal biochemical compositions and growth are greatly influenced by the availability of nutrients such as nitrogen and phosphorus, by which the limitation and depletion of nitrogen in the cultivation media can limit its growth with concurrent increases in lipid accumulation [3]. Nannochloropsis sp. is capable of accumulating triacylglycerols when exposed to nutrient limitation and responses to carbon dioxide fluctuation [4] particularly nitrate (N) and is therefore considered a promising organism for essential oil [5]. Moreover, Nannochloropsis sp. ...
Microalgae possess high oil content, exhibit rapid growth rates and biomass productivity, and leave a minimal environmental footprint, making them highly attractive for biofuel applications. Consequently, this present study is aimed at evaluating the impact of nitrogen starvation on the marine microalgae Nannochloropsis sp. to enhance lipid accumulation. The microalga culture was grown under an irradiance of 120 μmol photon m-2 s-1, temperature 25 ± 1 °C, continuous air-CO2, and with different concentrations of sodium nitrites over a 14-day cultivation period. From the results, it was found that the growth of the strain decreased under limited nitrogen conditions. After harvesting, the cells in the supernatant were regenerated by cultivating in double concentration of media. The results showed that on the 12th day, the highest lipid content was achieved, reaching 68.0 ± 2.3 wt.% of the dry weight under nitrogen limitation. The composition of the obtained lipid consisted of 18.0 wt.% saturated fatty acids, 72.38 wt.% monounsaturated fatty acids, and 8.95 wt.% polyunsaturated fatty acids. The lipid composition was dominated by monounsaturated oleic acid ( 70.97 ± 4.6 wt.%), saturated palmitic acid ( 13.68 ± 1.0 wt.%), and polyunsaturated linoleic acid ( 8.41 ± 0.4 wt.%). The findings of this study demonstrated that limiting nitrogen availability enhanced lipid accumulation in Nannochloropsis sp. Despite experiencing inhibited growth, this microalga species shows great potential for the large-scale cultivation of lipids and essential fatty acids. These results contribute to understanding the relationship between nitrogen availability and lipid metabolism in microalgae, providing valuable insights for optimizing lipid production in the context of biofuel and biotechnology applications.
Global warming is induced partly by rising atmospheric carbon dioxide levels, calling for sustainable methods to sequester carbon. Here we review carbon capture, usage, and storage with microalgae, with focus on methods to improve carbon
dioxide uptake, systems combining wastewater and flue gases, machine learning for strain identification, artificial intelligence and automation, and the circular bioeconomy. Carbon dioxide uptake by microalgae can be improved by using modified photobioreactors, membranes, chemical methods, solvents, adapted strains, genetically engineered strains, omics, and nanotechnology. We also discuss the economic viability of microalgae-based carbon capture and bioenergy generation. On an average, microalgal farming on 13 million acres area can sequester approximately 0.5 gigatons of CO2 to generate more than 300 tons of biomass.
Long noncoding RNAs exceeding a length of 200 nucleotides play an important role in ensuring cell functions and proper organism development by interacting with cellular compounds such as miRNA, mRNA, DNA and proteins. However, there is an additional level of lncRNA regulation, called lncRNA epigenetics, in gene expression control. In this review, we describe the most common modified nucleosides found in lncRNA, 6-methyladenosine, 5-methylcytidine, pseudouridine and inosine. The biosynthetic pathways of these nucleosides modified by the writer, eraser and reader enzymes are important to understanding these processes. The characteristics of the individual methylases, pseudouridine synthases and adenine–inosine editing enzymes and the methods of lncRNA epigenetics for the detection of modified nucleosides, as well as the advantages and disadvantages of these methods, are discussed in detail. The final sections are devoted to the role of modifications in the most abundant lncRNAs and their functions in pathogenic processes.
The first reference to the “C-value paradox” reported an apparent imbalance between organismal genome size and morphological complexity. Since then, next-generation sequencing has revolutionized genomic research and revealed that eukaryotic transcriptomes contain a large fraction of non-protein-coding components. Eukaryotic genomes are pervasively transcribed and noncoding regions give rise to a plethora of noncoding RNAs with undeniable biological functions. Among them, long noncoding RNAs (lncRNAs) seem to represent a new layer of gene expression regulation, participating in a wide range of molecular mechanisms at the transcriptional and post-transcriptional levels. In addition to their role in epigenetic regulation, plant lncRNAs have been associated with the degradation of complementary RNAs, the regulation of alternative splicing, protein sub-cellular localization, the promotion of translation and protein post-translational modifications. In this review, we report and integrate numerous and complex mechanisms through which long noncoding transcripts regulate post-transcriptional gene expression in plants.
Long non-coding RNAs (lncRNAs) are pervasive transcripts of longer than 200 nucleotides and indiscernible coding potential. lncRNAs are implicated as key regulatory molecules in various fundamental biological processes at transcriptional, post-transcriptional, and epigenetic levels. Advances in computational and experimental approaches have identified numerous lncRNAs in plants. lncRNAs have been found to act as prime mediators in plant growth, development, and tolerance to stresses. This review summarizes the current research status of lncRNAs in planta, their classification based on genomic context, their mechanism of action, and specific bioinformatics tools and resources for their identification and characterization. Our overarching goal is to summarize recent progress on understanding the regulatory role of lncRNAs in plant developmental processes such as flowering time, reproductive growth, and abiotic stresses. We also review the role of lncRNA in nutrient stress and the ability to improve biotic stress tolerance in plants. Given the pivotal role of lncRNAs in various biological processes, their functional characterization in agriculturally essential crop plants is crucial for bridging the gap between phenotype and genotype.
Long non-coding RNAs (lncRNAs) play critical regulatory roles in various biological processes. However, the presence of lncRNAs and how they function in plant polyploidy are still largely unknown. Hence, we examined the profile of lncRNAs in a nascent allotetraploid Cucumis hytivus (S14), its diploid parents, and the F1 hybrid, to reveal the function of lncRNAs in plant-interspecific hybridization and whole genome duplication. Results showed that 2206 lncRNAs evenly transcribed from all 19 chromosomes were identified in C. hytivus, 44.6% of which were from intergenic regions. Based on the expression trend in allopolyploidization, we found that a high proportion of lncRNAs (94.6%) showed up-regulated expression to varying degrees following hybridization. However, few lncRNAs (33, 2.1%) were non-additively expressed after genome duplication, suggesting the significant effect of hybridization on lncRNAs, rather than genome duplication. Furthermore, 253 cis-regulated target genes were predicted for these differentially expressed lncRNAs in S14, which mainly participated in chloroplast biological regulation (e.g., chlorophyll synthesis and light harvesting system). Overall, this study provides new insight into the function of lncRNAs during the processes of hybridization and polyploidization in plant evolution.
The unicellular industrial oleaginous microalgae Nannochloropsis spp. are model organisms for microalgal systems and synthetic biology. To facilitate community‐based annotation and mining of the rapidly accumulated functional genomics resources, we have initiated an international consortium and present a comprehensive, multi‐omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.cn). Via the Tripal toolkit, it featured user friendly interfaces hosting genomic resources with gene annotations, and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of N. oceanica IMET1 and N. salina CCMP 1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, genome browser, etc. were also included. In addition, functional validation of genes was indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources were also incorporated, especially for small RNA‐Seq including miRNAs and circRNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae.
Background
The immobile nature of plants means that they can be frequently confronted by various biotic and abiotic stresses during their lifecycle. Among the various abiotic stresses, water stress, temperature extremities, salinity, and heavy metal toxicity are the major abiotic stresses challenging overall plant growth. Plants have evolved complex molecular mechanisms to adapt under the given abiotic stresses. Long non-coding RNAs (lncRNAs)—a diverse class of RNAs that contain > 200 nucleotides(nt)—play an essential role in plant adaptation to various abiotic stresses.
Results
LncRNAs play a significant role as ‘biological regulators’ for various developmental processes and biotic and abiotic stress responses in animals and plants at the transcription, post-transcription, and epigenetic level, targeting various stress-responsive mRNAs, regulatory gene(s) encoding transcription factors, and numerous microRNAs (miRNAs) that regulate the expression of different genes. However, the mechanistic role of lncRNAs at the molecular level, and possible target gene(s) contributing to plant abiotic stress response and adaptation, remain largely unknown. Here, we review various types of lncRNAs found in different plant species, with a focus on understanding the complex molecular mechanisms that contribute to abiotic stress tolerance in plants. We start by discussing the biogenesis, type and function, phylogenetic relationships, and sequence conservation of lncRNAs. Next, we review the role of lncRNAs controlling various abiotic stresses, including drought, heat, cold, heavy metal toxicity, and nutrient deficiency, with relevant examples from various plant species. Lastly, we briefly discuss the various lncRNA databases and the role of bioinformatics for predicting the structural and functional annotation of novel lncRNAs.
Conclusions
Understanding the intricate molecular mechanisms of stress-responsive lncRNAs is in its infancy. The availability of a comprehensive atlas of lncRNAs across whole genomes in crop plants, coupled with a comprehensive understanding of the complex molecular mechanisms that regulate various abiotic stress responses, will enable us to use lncRNAs as potential biomarkers for tailoring abiotic stress-tolerant plants in the future.
Nutrient limited conditions are common in natural phytoplankton communities and are often used to increase the yield of lipids from industrial microalgae cultivations. Here we studied the effects of bioavailable nitrogen (N) and phosphorus (P) deprivation on the proteome and transcriptome of the oleaginous marine microalga Nannochloropsis gaditana. Turbidostat cultures were used to selectively apply either N or P deprivation, controlling for variables including the light intensity. Global (cell-wide) changes in the proteome were measured using Tandem Mass Tag (TMT) and LC-MS/MS, whilst gene transcript expression of the same samples was quantified by Illumina RNA-sequencing. We detected 3423 proteins, where 1543 and 113 proteins showed significant changes in abundance in N and P treatments, respectively. The analysis includes the global correlation between proteomic and transcriptomic data, the regulation of subcellular proteomes in different compartments, gene/protein functional groups, and metabolic pathways. The results show that triacylglycerol (TAG) accumulation under nitrogen deprivation was associated with substantial downregulation of protein synthesis and photosynthetic activity. Oil accumulation was also accompanied by a diverse set of responses including the upregulation of diacylglycerol acyltransferase (DGAT), lipase, and lipid body associated proteins. Deprivation of phosphorus had comparatively fewer, weaker effects, some of which were linked to the remodeling of respiratory metabolism.
The extraordinary maturation in high‐throughput sequencing technologies has revealed the existence of a complex network of transcripts in eukaryotic organisms, including thousands of long noncoding (lnc) RNAs with little or no protein‐coding capacity. Subsequent discoveries have shown that lncRNAs participate in a wide range of molecular processes, controlling gene expression and protein activity though direct interactions with proteins, DNA or other RNA molecules. Although significant advances have been achieved in the understanding of lncRNA biology in the animal kingdom, the functional characterization of plant lncRNAs is still in its infancy and remains a major challenge. In this review, we report emerging functional and mechanistic paradigms of plant lncRNAs and partner molecules, and discuss how cutting‐edge technologies may help to identify and classify yet uncharacterized transcripts into functional groups.
Often ignored and regarded as mere transcriptional noise, long noncoding RNAs (lncRNAs) are starting to be considered key regulators of gene expression across the Eukarya domain of life. In the model diatom Phaeodactylum tricornutum, we have previously reported the occurrence of 1,510 intergenic lncRNAs (lincRNAs), many of which displaying specific patterns of expression under phosphate fluctuation (Pi). Using strand-specific RNA-sequencing data we now expand the repertoire of P. tricornutum lncRNAs by identifying 2,628 novel natural antisense transcripts (NATs) that cover 21.5% of the annotated genomic loci. We found that NAT expression is tightly regulated by phosphate depletion and other naturally occurring environmental stresses. Furthermore, we identified 121 phosphate stress responsive NAT-mRNA pairs, the great majority of which showing a positive correlation (concordant pairs) and a small fraction with negative correlation (discordant pairs). Taken together our results show that NATs are highly abundant transcripts in P. tricornutum and that their expression is under tight regulation by nutrient and environmental stresses. Furthermore, our results suggest that in P. tricornutum Pi stress response NAT pairs predominantly regulate positively the expression of their cognate sense genes, the latter being involved in several biological processes underlying the control of cellular homeostasis under stress.
Genomic imprinting is a parent-of-origin dependent phenomenon that restricts transcription to predominantly one parental allele. Since the discovery of the first long noncoding RNA (lncRNA), which notably was an imprinted lncRNA, a body of knowledge has demonstrated pivotal roles for imprinted lncRNAs in regulating parental-specific expression of neighboring imprinted genes. In this Review, we will discuss the multiple functionalities attributed to lncRNAs and how they regulate imprinted gene expression. We also raise unresolved questions about imprinted lncRNA function, which may lead to new avenues of investigation. This Review is dedicated to the memory of Denise Barlow, a giant in the field of genomic imprinting and functional lncRNAs. With her passion for understanding the inner workings of science, her indominable spirit and her consummate curiosity, Denise blazed a path of scientific investigation that made many seminal contributions to genomic imprinting and the wider field of epigenetic regulation, in addition to inspiring future generations of scientists.
Background:
Long noncoding RNAs (lncRNAs) have several known functions involving various biological regulatory processes in plant. However, the possible roles of lncRNAs during peanut seed development have not been fully explored.
Results:
In this study, two peanut recombinant inbred lines (RIL8) that differ in seed size were used to investigate comprehensive lncRNA profiles derived from the seed development at 15 and 35 days after flowering (DAF). We identified a total of 9388 known and 4037 novel lncRNAs, from which 1437 were differentially expressed lncRNAs. Interestingly, the expression patterns of a number of lncRNAs can be very different between two closely related inbred lines and these lncRNAs were expressed predominantly in only one RIL at 35 DAF. Some differentially expressed lncRNAs were found related to putative cis-acting target genes and predicted to be involved in transcription, transport, cell division, and plant hormone biosynthesis. The expression patterns of several representative lncRNAs and 12 protein-coding genes were validated by qPCR. Same expression pattern was observed between most lncRNAs and their target genes. 11 lncRNAs, XR_001593099.1, MSTRG.18462.1, MSTRG.34915.1, MSTRG.41848.1, MSTRG.22884.1, MSTRG.12404.1, MSTRG.26719.1, MSTRG.35761.1, MSTRG.20033.1, MSTRG.13500.1, and MSTRG.9304.1 and their cis-acting target genes may play key roles in peanut seed development.
Conclusions:
These results provided new information on lncRNA-mediated regulatory roles in peanut seed development, contributing to the comprehensive understanding of the molecular mechanisms involved in peanut seed development.
Background:
Long non-coding RNAs (lncRNAs) play important roles in the regulation of plant responses to environmental stress by acting as essential regulators of gene expression. However, whether and how lncRNAs are involved in cold acclimation-dependent freezing tolerance in plants remains largely unknown. Medicago truncatula is a prominent model for studies of legume genomics, and distinguished by its cold-acclimation characteristics. To determine the roles of lncRNAs in plant cold stress response, we conducted genome-wide high-throughput sequencing in the legume model plant M. truncatula.
Results:
RNA-seq data were generated from twelve samples for the four treatments, i.e., non-cold treated leaves and roots, cold-treated leaves and roots of M. truncatula Jemalong A17 seedlings. A total of 1204 million raw reads were generated. Of them, 1150 million filtered reads after quality control (QC) were subjected to downstream analysis. A large number of 24,368 unique lncRNAs were identified from the twelve samples. Among these lncRNAs, 983 and 1288 were responsive to cold treatment in the leaves and roots, respectively. We further found that the intronic-lncRNAs were most sensitive to the cold treatment. The cold-responsive lncRNAs were unevenly distributed across the eight chromosomes in M. truncatula seedlings with obvious preferences for locations. Further analyses revealed that the cold-responsive lncRNAs differed between leaves and roots. The putative target genes of the lncRNAs were predicted to mainly involve the processes of protein translation, transport, metabolism and nucleic acid transcription. Furthermore, the networks of a tandem array of CBF/DREB1 genes that were reported to be located in a major freezing tolerance QTL region on chromosome 6 and their related lncRNAs were dissected based on their gene expression and chromosome location.
Conclusions:
We identified a comprehensive set of lncRNAs that were responsive to cold treatment in M. truncatula seedlings, and discovered tissue-specific cold-responsive lncRNAs in leaves and roots. We further dissected potential regulatory networks of CBF Intergenic RNA (MtCIR1) and MtCBFs that play critical roles in response and adaptation of M. truncatula to cold stress.
Nannochloropsis salina is a halotolerant, high-lipid-producing microalga that is being explored as a biofuel production species. Here, we report an improved high-quality draft assembly and annotation for the nuclear genome of N. salina strain CCMP1776.
Phytoplankton are primary producers in the marine ecosystem, where phosphorus is often a limiting factor of their growth. Hence, they have evolved strategies to recycle phosphorus by replacing membrane phospholipids with phosphorus-free lipids. However, mechanisms for replacement of lipid classes remain poorly understood. To improve our understanding, we performed the lipidomic and transcriptomic profiling analyses of an oleaginous marine microalga Nannochloropsis sp. PJ12 in response to phosphorus depletion (PD) and replenishing. In this study, by using (liquid chromatography couple with tandem mass spectrometry) LC-MS/MS-based lipidomic analysis, we show that membrane phospholipid levels are significantly reduced upon PD, while phosphorus-free betaine lipid levels are increased. However, levels of phosphorus-free photosynthetic galactolipid and sulfolipid are not increased upon PD, consistent with the reduced photosynthetic activity. RNA-seq-based transcriptomic analysis indicates that enzymes involved in phospholipid recycling and phosphorus-free lipid synthesis are upregulated, supporting the lipidomic analysis. Furthermore, enzymes involved in FASII (type II fatty acid synthesis) elongation cycle upon PD are transcriptionally downregulated. EPA (eicosapentaenoic acid) level decrease upon PD is revealed by both GC-MS (gas chromatography coupled with mass spectrometry) and LC-MS/MS-based lipidomic analyses. PD-induced alteration is reversed after phosphorus replenishing. Taken together, our results suggest that the alteration of lipid classes upon environmental change of phosphorus is a result of remodeling rather than de novo synthesis in Nannochloropsis sp. PJ12.
Brown algae have convergently evolved plant‐like body plans and reproductive cycles, which in plants are controlled by differential DNA methylation. This contribution provides the first single‐base methylome profiles of haploid gametophytes and diploid sporophytes of a multicellular alga.
Although only c. 1.4% of cytosines in Saccharina japonica were methylated mainly at CHH sites and characterized by 5‐methylcytosine (5mC), there were significant differences between life‐cycle stages. DNA methyltransferase 2 (DNMT2), known to efficiently catalyze tRNA methylation, is assumed to methylate the genome of S. japonica in the structural context of tRNAs as the genome does not encode any other DNA methyltransferases. Circular and long noncoding RNA genes were the most strongly methylated regulatory elements in S. japonica.
Differential expression of genes was negatively correlated with DNA methylation with the highest methylation levels measured in both haploid gametophytes. Hypomethylated and highly expressed genes in diploid sporophytes included genes involved in morphogenesis and halogen metabolism.
The data herein provide evidence that cytosine methylation, although occurring at a low level, is significantly contributing to the formation of different life‐cycle stages, tissue differentiation and metabolism in brown algae.
Phosphate availability is becoming a limiting environmental factor that inhibits plant growth and development. Here, we demonstrated that mutation of histone acetyltransferase GCN5 impaired phosphate-starvation responses (PSR) in Arabidopsis. Transcriptome analysis revealed that 888 GCN5-regulated candidate genes were potentially involved in responding to phosphate starvation. ChIP assay indicated that four genes, including a long non-coding RNA (lncRNA) At4, are direct targets of GCN5 in PSR regulation. In addition, GCN5-mediated H3K9/14 acetylation of At4 determined dynamic At4 expression. Consistent with the function of At4 in phosphate distribution, mutation of GCN5 impaired phosphate accumulation between shoots and roots under phosphate-deficiency condition, whereas constitutive expression of At4 in gcn5 mutants partially restored phosphate relocation. Further evidence proved that GCN5 regulation of At4 influenced the miRNA miR399 and its target PHO2 mRNA level. Taken together, we propose that GCN5-mediated histone acetylation plays a crucial role in PSR regulation via the At4-miR399-PHO2 pathway and provides a new epigenetic mechanism for the regulation of lncRNA in plants.
The species of the genus Nannochloropsis are unique in their maintenance of a nucleus-plastid continuum throughout their cell cycle, non-motility and asexual reproduction. These characteristics should have been endorsed in their gene assemblages (genomes). Here we show that N. oceanica has a genome of 29.3 Mb consisting of 32 pseudochromosomes and containing 7,330 protein-coding genes; and the host nucleus may have been overthrown by an ancient red alga symbiont nucleus during speciation through secondary endosymbiosis. In addition, N. oceanica has lost its flagella and abilities to undergo meiosis and sexual reproduction, and adopted a genome reduction strategy during speciation. We propose that N. oceanica emerged through the active fusion of a host protist and a photosynthesizing ancient red alga and the symbiont nucleus became dominant over the host nucleus while the chloroplast was wrapped by two layers of endoplasmic reticulum. Our findings evidenced an alternative speciation pathway of eukaryotes.
Background:
In industrial oleaginous microalgae such as Nannochloropsis spp., the key components of the carbon concentration mechanism (CCM) machineries are poorly defined, and how they are mobilized to facilitate cellular utilization of inorganic carbon remains elusive.
Results:
For Nannochloropsis oceanica, to unravel genes specifically induced by CO2 depletion which are thus potentially underpinning its CCMs, transcriptome, proteome and metabolome profiles were tracked over 0 h, 3 h, 6 h, 12 h and 24 h during cellular response from high CO2 level (HC; 50,000 ppm) to very low CO2 (VLC; 100 ppm). The activity of a biophysical CCM is evidenced based on induction of transcripts encoding a bicarbonate transporter and two carbonic anhydrases under VLC. Moreover, the presence of a potential biochemical CCM is supported by the upregulation of a number of key C4-like pathway enzymes in both protein abundance and enzymatic activity under VLC, consistent with a mitochondria-implicated C4-based CCM. Furthermore, a basal CCM underpinned by VLC-induced upregulation of photorespiration and downregulation of ornithine-citrulline shuttle and the ornithine urea cycles is likely present, which may be responsible for efficient recycling of mitochondrial CO2 for chloroplastic carbon fixation.
Conclusions:
Nannochloropsis oceanica appears to mobilize a comprehensive set of CCMs in response to very low CO2. Its genes induced by the stress are quite distinct from those of Chlamydomonas reinhardtii and Phaeodactylum tricornutum, suggesting tightly regulated yet rather unique CCMs. These findings can serve the first step toward rational engineering of the CCMs for enhanced carbon fixation and biomass productivity in industrial microalgae.
Understanding of the molecular responses underpinning diatom responses to ocean acidification is fundamental for predicting how important primary producers will be shaped by the continuous rise in atmospheric CO2. In this study, we have analyzed global transcriptomic changes of the model diatom Phaeodactylum tricornutum following growth for 15 generations in elevated pCO2 by strand-specific RNA sequencing (ssRNA-seq). Our results indicate that no significant effects of elevated pCO2 and associated carbonate chemistry changes on the physiological performance of the cells were observed after 15 generations whereas the expression of genes encoding histones and other genes involved in chromatin structure were significantly down-regulated, while the expression of transposable elements (TEs) and genes encoding histone acetylation enzymes were significantly up-regulated. Furthermore, we identified a series of long non-protein coding RNAs (lncRNAs) specifically responsive to elevated pCO2, suggesting putative regulatory roles for these largely uncharacterized genome components. Taken together, our integrative analyses reveal that epigenetic elements such as TEs, histone modifications and lncRNAs may have important roles in the acclimation of diatoms to elevated pCO2 over short time scales and thus may influence longer term adaptive processes in response to progressive ocean acidification.
Despite the many approaches to study differential splicing from RNA-seq, many challenges remain unsolved, including computing capacity and sequencing depth requirements. Here we present SUPPA2, a new method that addresses these challenges, and enables streamlined analysis across multiple conditions taking into account biological variability. Using experimental and simulated data, we show that SUPPA2 achieves higher accuracy compared to other methods, especially at low sequencing depth and short read length. We use SUPPA2 to identify novel Transformer2-regulated exons, novel microexons induced during differentiation of bipolar neurons, and novel intron retention events during erythroblast differentiation.
Electronic supplementary material
The online version of this article (10.1186/s13059-018-1417-1) contains supplementary material, which is available to authorized users.
Long non-coding RNAs (lncRNAs) have been defined as transcripts longer than 200 nucleotides, which lack significant protein coding potential and possess critical roles in diverse cellular processes. Long non-coding RNAs have recently been functionally characterized in plant stress–response mechanisms. In the present study, we perform a comprehensive identification of lncRNAs in response to combined stress induced by salinity and excess of boron in the Lluteño maize, a tolerant maize landrace from Atacama Desert, Chile. We use deep RNA sequencing to identify a set of 48,345 different lncRNAs, of which 28,012 (58.1%) are conserved with other maize (B73, Mo17 or Palomero), with the remaining 41.9% belonging to potentially Lluteño exclusive lncRNA transcripts. According to B73 maize reference genome sequence, most Lluteño lncRNAs correspond to intergenic transcripts. Interestingly, Lluteño lncRNAs presents an unusual overall higher expression compared to protein coding genes under exposure to stressed conditions. In total, we identified 1710 putatively responsive to the combined stressed conditions of salt and boron exposure. We also identified a set of 848 stress responsive potential trans natural antisense transcripts (trans-NAT) lncRNAs, which seems to be regulating genes associated with regulation of transcription, response to stress, response to abiotic stimulus and participating of the nicotianamine metabolic process. Reverse transcription-quantitative PCR (RT-qPCR) experiments were performed in a subset of lncRNAs, validating their existence and expression patterns. Our results suggest that a diverse set of maize lncRNAs from leaves and roots is responsive to combined salt and boron stress, being the first effort to identify lncRNAs from a maize landrace adapted to extreme conditions such as the Atacama Desert. The information generated is a starting point to understand the genomic adaptabilities suffered by this maize to surpass this extremely stressed environment.
Nannochloropsis is a genus of fast-growing microalgae that are regularly used for biotechnology applications. Nannochloropsis species have a high triacylglycerol content and their polar lipids are rich in the omega-3 long-chain polyunsaturated fatty acid, eicosapentaenoic acid. Placed in the heterokont lineage, the Nannochloropsis genus has a complex evolutionary history. Genome sequences are available for several species, and a number of transcriptomic datasets have been produced, making this genus a facile model for comparative genomics. There is a growing interest in Nannochloropsis species as models for the study of microalga lipid metabolism and as a chassis for synthetic biology. Recently, techniques for gene stacking, and targeted gene disruption and repression in the Nannochloropsis genus have been developed. These tools enable gene-specific, mechanistic studies and have already allowed the engineering of improved Nannochloropsis strains with superior growth, or greater bioproduction.
Long non-coding RNAs (lncRNAs) are a family of regulatory RNAs that play essential role in the various developmental processes and stress responses. Recent advances in sequencing technology and computational methods enabled identification and characterization of lncRNAs in certain plant species, but they are less known in Triticum aestivum (bread wheat). Herein, we analyzed 52 RNA seq data (>30 billion reads) and identified 44,698 lncRNAs in T. aestivum genome, which were characterized in comparison to the coding sequences (mRNAs). Similar to the mRNAs, lncRNAs were also derived from each sub-genome and chromosome, and showed tissue developmental stage specific and differential expression, as well. The modulated expression of lncRNAs during abiotic stresses like heat, drought, and salt indicated their putative role in stress response. The co-expression of lncRNAs with vital mRNAs including various transcription factors and enzymes involved in Abscisic acid (ABA) biosynthesis, and gene ontology mapping inferred their regulatory roles in numerous biological processes. A few lncRNAs were predicted as precursor (19 lncRNAs), while some as target mimics (1,047 lncRNAs) of known miRNAs involved in various regulatory functions. The results suggested numerous functions of lncRNAs in T. aestivum, and unfolded the opportunities for functional characterization of individual lncRNA in future studies.
Diatoms are eukaryotic microalgae that are responsible for up to 40% of the ocean’s primary productivity. How diatoms respond to environmental perturbations such as elevated carbon concentrations in the atmosphere is currently poorly understood. We developed a transcriptional regulatory network based on various transcriptome sequencing expression libraries for different environmental responses to gain insight into the marine diatom’s metabolic and regulatory interactions and provide a comprehensive framework of responses to increasing atmospheric carbon levels. This transcriptional regulatory network was integrated with a recently published genome-scale metabolic model of Phaeodactylum tricornutum to explore the connectivity of the regulatory network and shared metabolites. The integrated regulatory and metabolic model revealed highly connected modules within carbon and nitrogen metabolism. P. tricornutum ’s response to rising carbon levels was analyzed by using the recent genome-scale metabolic model with cross comparison to experimental manipulations of carbon dioxide.
IMPORTANCE Using a systems biology approach, we studied the response of the marine diatom Phaeodactylum tricornutum to changing atmospheric carbon concentrations on an ocean-wide scale. By integrating an available genome-scale metabolic model and a newly developed transcriptional regulatory network inferred from transcriptome sequencing expression data, we demonstrate that carbon metabolism and nitrogen metabolism are strongly connected and the genes involved are coregulated in this model diatom. These tight regulatory constraints could play a major role during the adaptation of P. tricornutum to increasing carbon levels. The transcriptional regulatory network developed can be further used to study the effects of different environmental perturbations on P. tricornutum ’s metabolism.
Microalgae are regarded as the most promising biofuel candidates and extensive metabolic engineering were conducted but very few improvements were achieved. Long non-coding RNA (lncRNA) investigation and manipulation may provide new insights for this issue. LncRNAs refer to transcripts that are longer than 200 nucleotides, do not encode proteins but play important roles in eukaryotic gene regulation. However, no information of potential lncRNAs has been reported in eukaryotic alga. Recently, we performed RNA sequencing in Chlamydomonas reinhardtii, and obtained totally 3,574 putative lncRNAs. 1440 were considered as high-confidence lncRNAs, including 936 large intergenic, 310 intronic and 194 anti-sense lncRNAs. The average transcript length, ORF length and numbers of exons for lncRNAs are much less than for genes in this green alga. In contrast with human lncRNAs of which more than 98% are spliced, the percentage in C. reinhardtii is only 48.1%. In addition, we identified 367 lncRNAs responsive to sulfur deprivation, including 36 photosynthesis-related lncRNAs. This is the first time that lncRNAs were explored in the unicellular model organism C. reinhardtii. The lncRNA data could also provide new insights into C. reinhardtii hydrogen production under sulfur deprivation.
Phosphorus (P) is an essential element to all living cells, yet fluctuations in P concentrations are recurrent in the marine environment. Diatoms are amongst the most successful phytoplankton groups, adapting to and surviving periods of suboptimal conditions and resuming growth as soon as nutrient concentrations permit. A knowledge of the molecular underpinnings of diatom ecological success is, however, still very incomplete.
By strand‐specific RNA sequencing, we analyzed the global transcriptome changes of the diatom Phaeodactylum tricornutum in response to P fluctuations over a course of 8 d, defining five distinct physiological states.
This study reports previously unidentified genes highly responsive to P stress in P. tricornutum . Our data also uncover the complexity of the P. tricornutum P‐responsive sensory and signaling system that combines bacterial two‐component systems with more complex pathways reminiscent of metazoans. Finally, we identify a multitude of novel long intergenic nonprotein coding RNA s (linc RNA s) specifically responsive to P depletion, suggesting putative regulatory roles in the regulation of P homeostasis.
Our work provides additional molecular insights into the resilience of diatoms and their ecological success, and opens up novel routes to address and explore the function and regulatory roles of P. tricornutum linc RNA s in the context of nutrient stress.
Microalgae are considered promising alternative energy sources because they consume CO 2 and accumulate large amounts of lipids that can be used as biofuel. Nannochloropsis is a particularly promising microalga due to its high growth rate and lipid content, and the availability of genomic information. Transcription factors (TFs) are global regulators of biological pathways by up- or down-regulation of related genes. Among these, basic helix-loop-helix (bHLH) TFs regulate growth, development, and stress responses in plants and animals, and have been identified in microalgae. We identified two bHLH TFs in the genome of N. salina CCMP1776, NsbHLH1, and NsbHLH2, and characterized functions of NsbHLH2 that may be involved in growth and nutrient uptake.
We obtained NsbHLH2 overexpressing transformants of N. salina CCMP1776 by particle bombardment and confirmed that these were stable transformants. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting using antibodies against the FLAG tag that was attached at the end of the coding sequence confirmed the expression of the NsbHLH2 protein under various culture conditions. The qRT-PCR results also indicated that the endogenous and transgenic expression of NsbHLH2 was reduced under stressed conditions. Overexpression of NsbHLH2 led to increased growth rate in the early growth period, and concomitantly higher nutrient uptake, than wild type (WT). These enhanced growth and nutrient uptake resulted in increased productivities of biomass and FAME. For example, one of the transformants, NsbHLH2 3–6, showed increased biomass productivity by 36 % under the normal condition, and FAME productivity by 33 % under nitrogen limitation condition. Conclusively, the improved growth in the transformants can be associated with the enhanced nutrient uptake. We are currently assessing their potential for scale-up cultivation with positive outcomes.
Overexpression of NsbHLH2 led to enhanced growth rate and nutrient uptake during the early growth phase, and increased biomass and FAME productivity, especially in the later period under normal and stressed conditions. Based on these results, we postulate that NsbHLH2 can be employed for the industrial production of biodiesel from N. salina.
Background
Long non-coding RNAs (lncRNAs) have been shown to play crucially regulatory roles in diverse biological processes involving complex mechanisms. However, information regarding the number, sequences, characteristics and potential functions of lncRNAs in plants is so far overly limited.
Results
Using high-throughput sequencing and bioinformatics analysis, we identified a total of 23,324 putative lncRNAs from control, osmotic stress- and salt stress-treated leaf and root samples of Medicago truncatula, a model legume species. Out of these lncRNAs, 7,863 and 5,561 lncRNAs were identified from osmotic stress-treated leaf and root samples, respectively. While, 7,361 and 7,874 lncRNAs were identified from salt stress-treated leaf and root samples, respectively. To reveal their potential functions, we analyzed Gene Ontology (GO) terms of genes that overlap with or are neighbors of the stress-responsive lncRNAs. Enrichments in GO terms in biological processes such as signal transduction, energy synthesis, molecule metabolism, detoxification, transcription and translation were found.
Conclusions
LncRNAs are likely involved in regulating plant’s responses and adaptation to osmotic and salt stresses in complex regulatory networks with protein-coding genes. These findings are of importance for our understanding of the potential roles of lncRNAs in responses of plants in general and M. truncatula in particular to abiotic stresses.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0530-5) contains supplementary material, which is available to authorized users.
Supplying microalgae with extra CO2 is a promising means for improving lipid production. The molecular mechanisms involved in lipid accumulation under conditions of elevated CO2, however, remain to be fully elucidated. To understand how elevated CO2 improves lipid production, we performed sequencing of Chlorella sorokiniana LS-2 cellular transcripts during growth and compared transcriptional dynamics of genes involved in carbon flow from CO2 to triacylglycerol. These analyses identified the majority genes of carbohydrate metabolism and lipid biosynthesis pathways in C. sorokiniana LS-2. Under high doses of CO2 , despite down-regulation of most de novo fatty acid biosynthesis genes, genes involved in carbohydrate metabolic pathways including carbon fixation, chloroplastic glycolysis, components of the pyruvate dehydrogenase complex (PDHC) and chloroplastic membrane transporters were upexpressed at the prolonged lipid accumulation phase. The data indicate that lipid production is largely independent of de novo fatty acid synthesis. Elevated CO2 might push cells to channel photosynthetic carbon precursors into fatty acid synthesis pathways, resulting in an increase of overall triacylglycerol generation. In support of this notion, genes involved in triacylglycerol biosynthesis were substantially up-regulated. Thus, elevated CO2 may influence regulatory dynamics and result in increased carbon flow to triacylglycerol, thereby providing a feasible approach to increase lipid production in microalgae.
© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
The acquisition and assimilation of inorganic carbon (Ci) represents the largest flux of inorganic matter in photosynthetic organisms; hence, this process is tightly regulated. We examined the Ci-dependent transcriptional and metabolic regulation in wild-type Synechocystis sp. PCC 6803 compared with a ∆ndhR mutant defective in the main transcriptional repressor for Ci acquisition genes, NdhR (CcmR). The analysis revealed that many protein-coding transcripts that are normally repressed in the presence of high CO2 concentrations (HC) were strongly expressed in ∆ndhR, whereas other mRNAs were strongly down-regulated in mutant cells, suggesting a potential activating role for NdhR. A conserved NdhR-binding motif was identified in the promoters of de-repressed genes. Interestingly, the expression of some NdhR-regulated genes remained further inducible under low CO2 conditions (LC), indicating the involvement of additional NdhR-independent Ci-regulatory mechanisms. Intriguingly, we also observed that the abundance of 52 antisense RNAs and 34 potential non-coding RNAs was affected by Ci supply, although most of these molecules were not regulated through NdhR. Thus, antisense and non-coding RNAs could contribute to NdhR-independent carbon regulation. In contrast to the transcriptome, the metabolome in ∆ndhR cells was similar to that of wild-type cells under HC conditions. This observation and the delayed metabolic responses to the LC-shift in ∆ndhR, specifically the lack of transient increases in the photorespiratory pathways intermediates 2-phosphoglycolate, glycolate and glycine, suggest that the deregulation of gene expression in the ∆ndhR mutant successfully pre-acclimates cyanobacterial cells to lowered Ci supply under HC conditions.
Copyright © 2015, American Society of Plant Biologists.
Alternative splicing is an essential mechanism for increasing transcriptome and proteome diversity in eukaryotes. Particularly in multicellular eukaryotes, this mechanism is involved in the regulation of developmental and physiological processes like growth, differentiation and signal transduction.
Here we report the genome-wide analysis of alternative splicing in the multicellular green alga Volvox carteri. The bioinformatic analysis of 132,038 expressed sequence tags (ESTs) identified 580 alternative splicing events in a total of 426 genes. The predominant type of alternative splicing in Volvox is intron retention (46.5%) followed by alternative 5′ (17.9%) and 3′ (21.9%) splice sites and exon skipping (9.5%). Our analysis shows that in Volvox at least ~2.9% of the intron-containing genes are subject to alternative splicing. Considering the total number of sequenced ESTs, the Volvox genome seems to provide more favorable conditions (e.g., regarding length and GC content of introns) for the occurrence of alternative splicing than the genome of its close unicellular relative Chlamydomonas. Moreover, many randomly chosen alternatively spliced genes of Volvox do not show alternative splicing in Chlamydomonas. Since the Volvox genome contains about the same number of protein-coding genes as the Chlamydomonas genome (~14,500 protein-coding genes), we assumed that alternative splicing may play a key role in generation of genomic diversity, which is required to evolve from a simple one-cell ancestor to a multicellular organism with differentiated cell types (Mol Biol Evol 31:1402-1413, 2014). To confirm the alternative splicing events identified by bioinformatic analysis, several genes with different types of alternatively splicing have been selected followed by experimental verification of the predicted splice variants by RT-PCR.
The results show that our approach for prediction of alternative splicing events in Volvox was accurate and reliable. Moreover, quantitative real-time RT-PCR appears to be useful in Volvox for analyses of relationships between the appearance of specific alternative splicing variants and different kinds of physiological, metabolic and developmental processes as well as responses to environmental changes.
Background
Long noncoding RNAs (lncRNAs) play important roles in a wide range of biological processes in mammals and plants. However, the systematic examination of lncRNAs in plants lags behind that in mammals. Recently, lncRNAs have been identified in Arabidopsis and wheat; however, no systematic screening of potential lncRNAs has been reported for the rice genome.
Results
In this study, we perform whole transcriptome strand-specific RNA sequencing (ssRNA-seq) of samples from rice anthers, pistils, and seeds 5 days after pollination and from shoots 14 days after germination. Using these data, together with 40 available rice RNA-seq datasets, we systematically analyze rice lncRNAs and definitively identify lncRNAs that are involved in the reproductive process. The results show that rice lncRNAs have some different characteristics compared to those of Arabidopsis and mammals and are expressed in a highly tissue-specific or stage-specific manner. We further verify the functions of a set of lncRNAs that are preferentially expressed in reproductive stages and identify several lncRNAs as competing endogenous RNAs (ceRNAs), which sequester miR160 or miR164 in a type of target mimicry. More importantly, one lncRNA, XLOC_057324, is demonstrated to play a role in panicle development and fertility. We also develop a source of rice lncRNA-associated insertional mutants.
Conclusions
Genome-wide screening and functional analysis enabled the identification of a set of lncRNAs that are involved in the sexual reproduction of rice. The results also provide a source of lncRNAs and associated insertional mutants in rice.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0512-1) contains supplementary material, which is available to authorized users.
To reveal the molecular mechanisms of oleaginousness in microalgae, transcriptomic and lipidomic dynamics of the oleaginous microalga Nannochloropsis oceanica IMET1 under nitrogen-replete (N+) and N-depleted (N-) conditions were simultaneously tracked. At the transcript level, enhanced triacylglycerol (TAG) synthesis under N- conditions primarily involved upregulation of seven putative diacylglycerol acyltransferase (DGAT) genes and downregulation of six other DGAT genes, with a simultaneous elevation of the other Kennedy pathway genes. Under N- conditions, despite downregulation of most de novo fatty acid synthesis genes, the pathways that shunt carbon precursors from protein and carbohydrate metabolic pathways into glycerolipid synthesis were stimulated at the transcript level. In particular, the genes involved in supplying carbon precursors and energy for de novo fatty acid synthesis, including those encoding components of the pyruvate dehydrogenase complex (PDHC), glycolysis, and PDHC bypass, and suites of specific transporters, were substantially upregulated under N- conditions, resulting in increased overall TAG production. Moreover, genes involved in the citric acid cycle and β-oxidation in mitochondria were greatly enhanced to utilize the carbon skeletons derived from membrane lipids and proteins to produce additional TAG or its precursors. This temporal and spatial regulation model of oil accumulation in microalgae provides a basis for improving our understanding of TAG synthesis in microalgae and will also enable more rational genetic engineering of TAG production.
Alternative splicing (AS) is common in higher eukaryotes and plays an important role in gene posttranscriptional regulation. It has been suggested that AS varies dramatically among species, tissues, and duplicated gene families of different sizes. However, the genomic forces that govern AS variation remain poorly understood. Here, through genome-wide identification of AS events in the soybean (Glycine max) genome using high-throughput RNA sequencing of 28 samples from different developmental stages, we found that more than 63% of multiexonic genes underwent AS. More AS events occurred in the younger developmental stages than in the older developmental stages for the same type of tissue, and the four main AS types, exon skipping, intron retention, alternative donor sites, and alternative acceptor sites, exhibited different characteristics. Global computational analysis demonstrated that the variations of AS frequency and AS types were significantly correlated with the changes of gene features and gene transcriptional level. Further investigation suggested that the decrease of AS within the genome-wide duplicated genes were due to the diminution of intron length, exon number, and transcriptional level. Altogether, our study revealed that a large number of genes were alternatively spliced in the soybean genome and that variations in gene structure and transcriptional level may play important roles in regulating AS.
Oleaginous microalgae are promising feedstock for biofuels, yet the genetic diversity, origin and evolution of oleaginous traits remain largely unknown. Here we present a detailed phylogenomic analysis of five oleaginous Nannochloropsis species (a total of six strains) and one time-series transcriptome dataset for triacylglycerol (TAG) synthesis on one representative strain. Despite small genome sizes, high coding potential and relative paucity of mobile elements, the genomes feature small cores of ca. 2,700 protein-coding genes and a large pan-genome of >38,000 genes. The six genomes share key oleaginous traits, such as the enrichment of selected lipid biosynthesis genes and certain glycoside hydrolase genes that potentially shift carbon flux from chrysolaminaran to TAG synthesis. The eleven type II diacylglycerol acyltransferase genes (DGAT-2) in every strain, each expressed during TAG synthesis, likely originated from three ancient genomes, including the secondary endosymbiosis host and the engulfed green and red algae. Horizontal gene transfers were inferred in most lipid synthesis nodes with expanded gene doses and many glycoside hydrolase genes. Thus multiple genome pooling and horizontal genetic exchange, together with selective inheritance of lipid synthesis genes and species-specific gene loss, have led to the enormous genetic apparatus for oleaginousness and the wide genomic divergence among present-day Nannochloropsis. These findings have important implications in the screening and genetic engineering of microalgae for biofuels.
Recent research on long noncoding RNAs (lncRNAs) has expanded our understanding of gene transcription regulation and the generation of cellular complexity. Depending on their genomic origins, lncRNAs can be transcribed from intergenic or intragenic regions or from introns of protein coding genes. We have recently reported more than 6,000 intergenic lncRNAs in Arabidopsis. Here, we systematically identified long noncoding natural antisense transcripts (lncNATs), defined as lncRNAs transcribed from the opposite DNA strand of coding or noncoding genes. We found a total of 37,238 sense-antisense transcript pairs and 70% of annotated mRNAs to be associated with antisense transcripts in Arabidopsis. These lncNATs could be reproducibly detected by different technical platforms, including strand-specific tiling arrays, Agilent custom expression arrays, strand-specific RNA-seq and qRT-PCR experiments. Moreover, we investigated the expression profiles of sense-antisense pairs in response to light and observed spatial and developmental-specific light effects on 626 concordant and 766 discordant NAT pairs. Genes for a large number of the light-responsive NAT pairs are associated with histone modification peaks, and histone acetylation is dynamically correlated with light-responsive expression changes of NATs.
Nannochloropsis is rapidly emerging as a model organism for the study of biofuel production in microalgae.Here we report a high quality genomic assembly of Nannochloropsis gaditana, consisting of large contigs, up to 500 kbp long, and scaffolds that in most cases span the entire length of chromosomes. We identified 10,646 complete genes and characterized possible alternative transcripts. The annotation of the predicted genes and the analysis of cellular processes revealed traits relevant for the genetic improvement of this organism such as genes involved in DNA recombination, RNA silencing and cell wall synthesis.We also analysed the modification of the transcriptional profile in nitrogen deficiency, a condition known to stimulate lipid accumulation. While the content of lipids increased, we did not detect major changes in expression of the genes involved in their biosynthesis. At the same time we observed a very significant down regulation of mitochondrial gene expression, suggesting that part of the Acetyl-CoA and NAD(P)H, normally oxidized through the mitochondrial respiration, would be made available for fatty acids synthesis, increasing the flux through the lipid biosynthetic pathway.Finally we released an information resource of the genomic data of N. gaditana, available at www.nannochloropsis.org.
Microalgae are promising feedstock for production of lipids, sugars, bioactive compounds and in particular biofuels, yet development of sensitive and reliable phylotyping strategies for microalgae has been hindered by the paucity of phylogenetically closely-related finished genomes.
Using the oleaginous eustigmatophyte Nannochloropsis as a model, we assessed current intragenus phylotyping strategies by producing the complete plastid (pt) and mitochondrial (mt) genomes of seven strains from six Nannochloropsis species. Genes on the pt and mt genomes have been highly conserved in content, size and order, strongly negatively selected and evolving at a rate 33% and 66% of nuclear genomes respectively. Pt genome diversification was driven by asymmetric evolution of two inverted repeats (IRa and IRb): psbV and clpC in IRb are highly conserved whereas their counterparts in IRa exhibit three lineage-associated types of structural polymorphism via duplication or disruption of whole or partial genes. In the mt genomes, however, a single evolution hotspot varies in copy-number of a 3.5Kb-long, cox1-harboring repeat. The organelle markers (e.g., cox1, cox2, psbA, rbcL and rrn16_mt) and nuclear markers (e.g., ITS2 and 18S) that are widely used for phylogenetic analysis obtained a divergent phylogeny for the seven strains, largely due to low SNP density. A new strategy for intragenus phylotyping of microalgae was thus proposed that includes (i) twelve sequence markers that are of higher sensitivity than ITS2 for interspecies phylogenetic analysis, (ii) multi-locus sequence typing based on rps11_mt-nad4, rps3_mt and cox2-rrn16_mt for intraspecies phylogenetic reconstruction and (iii) several SSR loci for identification of strains within a given species.
This first comprehensive dataset of organelle genomes for a microalgal genus enabled exhaustive assessment and searches of all candidate phylogenetic markers on the organelle genomes. A new strategy for intragenus phylotyping of microalgae was proposed which may be generally applicable to other microalgal genera and should serve as a valuable tool in the expanding algal biotechnology industry.
TopHat is a popular spliced aligner for RNA-seq experiments. Here, we describe TopHat2, which incorporates many significant enhancements to TopHat. TopHat2 can align reads of various lengths produced by the latest sequencing technologies, while allowing for variable-length indels with respect to the reference genome. In addition to de novo spliced alignment, TopHat2 can align reads across fusion breaks, which occur after genomic translocations. TopHat2 combines the ability to discover novel splice sites with direct mapping to known transcripts, producing sensitive and accurate alignments, even for highly repetitive genomes or in the presence of pseudogenes. TopHat2 is available at http://ccb.jhu.edu/software/tophat.
Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.
Rising CO 2 concentrations and their effects on plant productivity present challenging issues. Effects on the photosynthesis/photorespiration balance and changes in primary metabolism are known, caused by the competitive interaction of CO 2 and O 2 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase. However, impacts on stress resistance are less clear. Reactive oxygen species are key players in biotic and abiotic stress responses, but there is no consensus on whether elevated CO 2 constitutes a stress. Although high CO 2 increases yield in C 3 plants, it can also increase cellular oxidation and activate phytohormone defense pathways. Reduction-oxidation processes play key roles in acclimation to high CO 2 , with specific enzymes acting in compartment-specific signaling. Traditionally, acclimation to high CO 2 has been considered in terms of altered carbon gain, but emerging evidence suggests that CO 2 is a signal as well as a substrate. Some CO 2 effects on defense are likely mediated independently of primary metabolism. Nonetheless, primary photosynthetic metabolism is highly integrated with defense and stress signaling pathways, meaning that plants will be able to acclimate to the changing environment over the coming decades.
Photosynthetic organisms need to respond frequently to the fluctuation of light quality and light quantity in their habitat. In response to the fluctuation of different single wavelength lights, these organisms have to adjust and optimize the employment of light energy by redistributing excitation energy and remodeling photosystem stoichiometry or light complex structure. However, the response of whole cellular processes to fluctuations in single wavelength light is mostly unknown. Here, we report the transcriptomic and proteomic dynamics and metabolic adaptation mechanisms of Nannochloropsis oceanica to blue and red light. Preferential exposure to different light spectra induces massive reprogramming of the Nannochloropsis transcriptome and proteome. Combined with physiological and biochemical investigation, the rewiring of many cellular processes was observed, including carbon/nitrogen assimilation, photosynthesis, chlorophyll and cartenoid biosynthesis, reactive oxygen species (ROS) scavenging systems, and chromatin state regulation. A strong and rapid regulation of genes or proteins related to nitrogen metabolism, photosynthesis, chlorophyll synthesis, ROS scavenging system, and carotenoid metabolism were observed during 12 h and 24 h of exposure under red light. Additionally, two light harvesting complex proteins induced by blue light and one by red light were observed. The differential responses of N. oceanica to red and blue irradiation reveal how marine microalgae adapt to change in light quality and can be exploited for biofuel feedstock development.
Over the last decade, it has been increasingly demonstrated that the genomes of many species are pervasively transcribed, resulting in the production of numerous long noncoding RNAs (lncRNAs). At the same time, it is now appreciated that many types of DNA regulatory elements, such as enhancers and promoters, regularly initiate bi-directional transcription. Thus, discerning functional noncoding transcripts from a vast transcriptome is a paramount priority, and challenge, for the lncRNA field. In this review, we aim to provide a conceptual and experimental framework for classifying and elucidating lncRNA function. We categorize lncRNA loci into those that regulate gene expression in cis versus those that perform functions in trans and propose an experimental approach to dissect lncRNA activity based on these classifications. These strategies to further understand lncRNAs promise to reveal new and unanticipated biology with great potential to advance our understanding of normal physiology and disease.
Microalgae have been studied as biofactories for almost four decades. Yet, even until today, many aspects of microalgae farming and processing are still considered exploratory because of the uniqueness of each microalgal species. Thus, it is important to develop the entire process of microalgae farming: from culturing to harvesting, and down to extracting the desired high-value products. Based on its rapid growth and high oil productivities, Nannochloropsis sp. is of particular interest to many industries for the production of high-value oil containing omega-3 fatty acids, specifically eicosapentaenoic acid (EPA), but also several other products. This review compares the various techniques for induction, harvesting, and extraction of EPA-rich oil and high-value protein explored by academia and industry to develop a multi-product Nannochloropsis biorefinery. Knowledge gaps and opportunities are discussed for culturing and inducing fatty acid biosynthesis, biomass harvesting, and extracting EPA-rich oil and high-value protein from the biomass of Nannochloropsis sp.
The genome of the filamentous brown alga Ectocarpus was the first to be completely sequenced from within the brown algal group and has served as a key reference genome both for this lineage and for the stramenopiles.
We present a complete structural and functional reannotation of the Ectocarpus genome.
The large‐scale assembly of the Ectocarpus genome was significantly improved and genome‐wide gene re‐annotation using extensive RNA ‐seq data improved the structure of 11 108 existing protein‐coding genes and added 2030 new loci. A genome‐wide analysis of splicing isoforms identified an average of 1.6 transcripts per locus. A large number of previously undescribed noncoding genes were identified and annotated, including 717 loci that produce long noncoding RNA s. Conservation of lnc RNA s between Ectocarpus and another brown alga, the kelp Saccharina japonica , suggests that at least a proportion of these loci serve a function. Finally, a large collection of single nucleotide polymorphism‐based markers was developed for genetic analyses. These resources are available through an updated and improved genome database.
This study significantly improves the utility of the Ectocarpus genome as a high‐quality reference for the study of many important aspects of brown algal biology and as a reference for genomic analyses across the stramenopiles.
High-throughput sequencing of mRNA (RNA-seq) has become the standard method for measuring and comparing the levels of gene expression in a wide variety of species and conditions. RNA-seq experiments generate very large, complex data sets that demand fast, accurate and flexible software to reduce the raw read data to comprehensible results. HISAT (hierarchical indexing for spliced alignment of transcripts), StringTie and Ballgown are free, open-source software tools for comprehensive analysis of RNA-seq experiments. Together, they allow scientists to align reads to a genome, assemble transcripts including novel splice variants, compute the abundance of these transcripts in each sample and compare experiments to identify differentially expressed genes and transcripts. This protocol describes all the steps necessary to process a large set of raw sequencing reads and create lists of gene transcripts, expression levels, and differentially expressed genes and transcripts. The protocol's execution time depends on the computing resources, but it typically takes under 45 min of computer time. HISAT, StringTie and Ballgown are available from http://ccb.jhu.edu/software.shtml.
Long noncoding RNAs (lncRNAs) play important roles in various biological regulatory processes in yeast, mammals, and plants. However, no systematic identification of lncRNAs has been reported in Gossypium arboreum. In this study, the strand-specific RNA sequencing (ssRNA-seq) of samples from cotton fibers and leaves was performed, and lncRNAs involved in fiber initiation and elongation processes were systematically identified and analyzed. We identified 5,996 lncRNAs, of which 3,510 and 2,486 can be classified as long intergenic noncoding RNAs (lincRNAs) and natural antisense transcripts (lncNAT), respectively. LincRNAs and lncNATs are similar in many aspects, but have some differences in exon number, exon length, and transcript length. Expression analysis revealed that 51.9% of lincRNAs and 54.5% of lncNATs transcripts were preferentially expressed at one stage of fiber development, and were significantly highly expressed than protein-coding transcripts (21.7%). During the fiber and rapid elongation stages, rapid and dynamic changes in lncRNAs may contribute to fiber development in cotton. This work describes a set of lncRNAs that are involved in fiber development. The characterization and expression analysis of lncRNAs will facilitate future studies on their roles in fiber development in cotton.
Nannochloropsis oceanica CCMP1779 is a marine unicellular stramenopile and an emerging reference species for basic research on oleogenic microalgae with biotechnological relevance. We investigated its physiology and transcriptome under light/dark cycles. We observed oscillations in lipid content and a predominance of cell division in the first half of the dark phase. Globally, more than 60% of the genes cycled in N. oceanica CCMP1779, with gene expression peaking at different times of the day. Interestingly, the phase of expression of genes involved in certain biological processes was conserved across photosynthetic lineages. Furthermore, in agreement with our physiological studies we found the processes of lipid metabolism and cell division enriched in cycling genes. For example, there was tight coordination of genes involved in the lower part of glycolysis, fatty acid synthesis and lipid production at dawn preceding lipid accumulation during the day. Our results suggest that diel lipid storage plays a key role for N. oceanica CCMP1779 growth under natural conditions making this alga a promising model to gain a basic mechanistic understanding of triacylglycerol production in photosynthetic cells. Our data will help the formulation of new hypotheses on the role of cyclic gene expression in cell growth and metabolism in Nannochloropsis. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Short noncoding RNA s have been demonstrated to play important roles in regulation of gene expression and stress responses, but the repertoire and functions of long noncoding RNA s ( l nc RNA s) remain largely unexplored, particularly in plants.
To explore the role of l nc RNA s in disease resistance, we used a strand‐specific RNA ‐sequencing approach to identify l nc RNA s responsive to F usarium oxysporum infection in A rabidopsis thaliana .
Antisense transcription was found in c . 20% of the annotated A . thaliana genes. Several noncoding natural antisense transcripts responsive to F . oxysporum infection were found in genes implicated in disease defense. While the majority of the novel transcriptionally active regions ( TAR s) were adjacent to annotated genes and could be an extension of the annotated transcripts, 159 novel intergenic TAR s, including 20 F . oxysporum ‐responsive l nc TAR s, were identified. Ten F . oxysporum ‐induced l nc TAR s were functionally characterized using T ‐ DNA insertion or RNA ‐interference knockdown lines, and five were demonstrated to be related to disease development. Promoter analysis suggests that some of the F . oxysporum ‐induced l nc TAR s are direct targets of transcription factor(s) responsive to pathogen attack.
Our results demonstrated that strand‐specific RNA sequencing is a powerful tool for uncovering hidden levels of transcriptome and that Inc RNA s are important components of the antifungal networks in A . thaliana .
The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. The discovery of extensive transcription of large RNA transcripts that do not code for proteins, termed long noncoding RNAs (lncRNAs), provides an important new perspective on the centrality of RNA in gene regulation. Here, we discuss genome-scale strategies to discover and characterize lncRNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators and then target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, lncRNAs can function as modular scaffolds to specify higher-order organization in RNP complexes and in chromatin states. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs for proper gene control across all kingdoms of life.