ceRNA crosstalk mediated by ncRNAs is a novel regulatory mechanism in fish sex determination and differentiation

Competing endogenous RNAs (ceRNAs) are vital regulators of gene networks in mammals. The involvement of noncoding RNAs (ncRNAs) as ceRNA in genotypic sex determination (GSD) and environmental sex determination (ESD) in fish is unknown. The Chinese tongue sole, which has both GSD and ESD mechanisms, was used to map the dynamic expression pattern of ncRNAs and mRNA in gonads during sex determination and differentiation. Transcript expression patterns shift during the sex differentiation phase, and ceRNA modulation occurs through crosstalk of differentially expressed long ncRNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and sex-related genes in fish. Of note was the significant up-regulation of a circRNA from the sex-determining gene dmrt1 (circular RNA dmrt1) and a lncRNA, called AMSDT (which stands for associated with male sex differentiation of tongue sole) in Chinese tongue sole testis. These two ncRNAs both share the same miRNA response elements with gsdf, which has an up-regulated expression when they bind to miRNA cse-miR-196 and concurrent down-regulated female sex-related genes to facilitate testis differentiation. This is the first demonstration in fish that ceRNA crosstalk mediated by ncRNAs modulates sexual development and unveils a novel regulatory mechanism for sex determination and differentiation.info:eu-repo/semantics/publishedVersio

Proteomic study uncovers molecular principles of single-cell-level phenotypic heterogeneity in lipid storage of Nannochloropsis oceanica

Abstract Background Nannochloropsis oceanica belongs to a large group of photoautotrophic eukaryotic organisms that play important roles in fixation and cycling of atmospheric CO2. Its capability of storing solar energy and carbon dioxide in the form of triacylglycerol (TAG) of up to 60% of total weight under nitrogen deprivation stress sparked interest in its use for biofuel production. Phenotypes varying in lipid accumulation among an N. oceanica population can be disclosed by single-cell analysis/sorting using fluorescence-activated cell sorting (FACS); yet the phenomenon of single cell heterogeneity in an algae population remains to be fully understood at the molecular level. In this study, combination of FACS and proteomics was used for identification, quantification and differentiation of these heterogeneities on the molecular level. Results For N. oceanica cultivated under nitrogen deplete (−N) and replete (+N) conditions, two groups differing in lipid content were distinguished. These differentiations could be recognized on the population as well as the single-cell levels; proteomics uncovered alterations in carbon fixation and flux, photosynthetic machinery, lipid storage and turnover in the populations. Although heterogeneity patterns have been affected by nitrogen supply and cultivation conditions of the N. oceanica populations, differentiation itself seems to be very robust against these factors: cultivation under +N, −N, in shaker bottles, and in a photo-bioreactor all split into two subpopulations. Intriguingly, population heterogeneity resumed after subpopulations were separately recultivated for a second round, refuting the possible development of genetic heterogeneity in the course of sorting and cultivation. Conclusions This work illustrates for the first time the feasibility of combining FACS and (prote)-omics for mechanistic understanding of phenotypic heterogeneity in lipid-producing microalgae. Such combinatorial method can facilitate molecular breeding and design of bioprocesses

Integration of proteome and transcriptome refines key molecular processes underlying oil production in Nannochloropsis oceanica\textit {Nannochloropsis oceanica}

$\bf Background$ Under nitrogen deficiency situation, Nannochloropsis spp. accumulate large amounts of lipids in the form of triacylglycerides (TAG). Mechanisms of this process from the perspective of transcriptome and metabolome have been obtained previously, yet proteome analysis is still sparse which hinders the analysis of dynamic adaption to nitrogen deficiency. Here, proteomes for 3 h, 6 h, 12 h, 24 h, 48 h and 10th day of nitrogen deplete (N−) and replete (N+) conditions were obtained and integrated with previous transcriptome data for $\textit {N. oceanica}$. $\bf Results$ Physiological adaptations to N− not apparent from transcriptome data were unveiled: (a) abundance of proteins related to photosynthesis only slightly decreased in the first 48 h, indicating that photosynthesis is still working efficiently, and protein amounts adjust gradually with reduction in chloroplast size. (b) Most proteins related to the TCA cycle were strongly upregulated after 48 h under N−, suggesting that respiration is enhanced after 48 h and that TCA cycle efflux supports the carbon required for lipid synthesis. (c) Proteins related to lipid accumulation via the Kennedy pathway increased their abundance at 48 h, synchronous with the previously reported diversification of fatty acids after 48 h. $\bf Conclusions$ This study adds a proteome perspective on the major pathways for TAG accumulation in $\textit {Nannochloropsis spp}$. Temporal changes of proteome exhibited distinct adaptation phases that are usually delayed relative to transcriptomic responses. Notably, proteome data revealed that photosynthesis and carbon fixation are still ongoing even after 48 h of N−. Moreover, sometimes completely opposite trends in proteome and transcriptome demonstrate the relevance of underexplored post-transcriptional regulation for N− adaptation

Transcriptomic and proteomic responses to very low CO2 suggest multiple carbon concentrating mechanisms in Nannochloropsis oceanica

AbstractBackgroundIn 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.ResultsFor 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.ConclusionsNannochloropsis 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

Knockdown of carbonate anhydrase elevates Nannochloropsis productivity at high CO2 level

Improving acid tolerance is pivotal to the development of microalgal feedstock for converting flue gas to biomass or oils. In the industrial oleaginous microalga Nannochloropsis oceanica, transcript knockdown of a cytosolic carbonic anhydrase (CA2), which is a key Carbon Concentrating Mechanism (CCM) component induced under 100 ppm CO2 (very low carbon, or VLC), results in similar to 45%, similar to 30% and similar to 40% elevation of photosynthetic oxygen evolution rate, growth rate and biomass accumulation rate respectively under 5% CO2 (high carbon, or HC), as compared to the wild type. Such high-CO2-level activated biomass over-production is reproducible across photobioreactor types and cultivation scales. Transcriptomic, proteomic and physiological changes of the mutant under high CO2 (HC; 5% CO2) suggest a mechanism where the higher pH tolerance is coupled to reduced biophysical CCM, sustained pH hemostasis, stimulated energy intake and enhanced photosynthesis. Thus &quot;inactivation of CCM&quot; can generate hyper-CO2-assimilating and autonomously containable industrial microalgae for flue gas-based oil production.</p

The NanDeSyn database for Nannochloropsis systems and synthetic biology

Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; ). Via the Tripal toolkit, it features user-friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. 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