Keeping time in the dark: Potato diel and circadian rhythmic gene expression reveals tissue-specific circadian clocks.

The circadian clock is an internal molecular oscillator and coordinates numerous physiological processes through regulation of molecular pathways. Tissue-specific clocks connected by mobile signals have previously been found to run at different speeds in Arabidopsis thaliana tissues. However, tissue variation in circadian clocks in crop species is unknown. In this study, leaf and tuber global gene expression in cultivated potato under cycling and constant environmental conditions was profiled. In addition, we used a circadian-regulated luciferase reporter construct to study tuber gene expression rhythms. Diel and circadian expression patterns were present among 17.9% and 5.6% of the expressed genes in the tuber. Over 500 genes displayed differential tissue specific diel phases. Intriguingly, few core circadian clock genes had circadian expression patterns, while all such genes were circadian rhythmic in cultivated tomato leaves. Furthermore, robust diel and circadian transcriptional rhythms were observed among detached tubers. Our results suggest alternative regulatory mechanisms and/or clock composition is present in potato, as well as the presence of tissue-specific independent circadian clocks. We have provided the first evidence of a functional circadian clock in below-ground storage organs, holding important implications for other storage root and tuberous crops.

Aureochromes maintain polyunsaturated fatty acid content in Nannochloropsis oceanica.

Nannochloropsis oceanica, like other stramenopile microalgae, is rich in long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA). We observed that fatty acid desaturases (FADs) involved in LC-PUFA biosynthesis were among the strongest blue light-induced genes in N. oceanica CCMP1779. Blue light was also necessary for maintaining LC-PUFA levels in CCMP1779 cells, and growth under red light led to a reduction in EPA content. Aureochromes are stramenopile-specific proteins that contain a light-oxygen-voltage (LOV)-sensing domain that associates with a flavin mononucleotide and is able to sense blue light. These proteins also contain a basic leucine zipper DNA-binding motif and can act as blue light-regulated transcription factors by associating with an E-box like motif, which we found enriched in the promoters of blue light-induced genes. We demonstrated that, in vitro, two CCMP1779 aureochromes were able to absorb blue light. Moreover, the loss or reduction of the expression of any of the three aureochrome genes led to a decrease in the blue light-specific induction of several FADs in CCMP1779. EPA content was also significantly reduced in NoAUREO2 and NoAUREO4 mutants. Taken together, our results indicate that aureochromes mediate blue light-dependent regulation of LC-PUFA content in N. oceanica CCMP1779 cells.

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; http://nandesyn.single-cell.cn). 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.

The brown clock: circadian rhythms in stramenopiles.

Circadian clocks allow organisms to anticipate environmental changes associated with the diurnal light/dark cycle. Circadian oscillators have been described in plants and green algae, cyanobacteria, animals and fungi, however, little is known about the circadian clocks of photosynthetic eukaryotes outside the green lineage. Stramenopiles are a diverse group of secondary endosymbionts whose plastid originated from a red alga. Photosynthetic stramenopiles, which include diatoms and brown algae, play key roles in biogeochemical cycles and are important components of marine ecosystems. Genome annotation efforts indicated the presence of a novel type of oscillator in these organisms and the first circadian clock component in a stramenopile has been recently discovered. This review summarizes the phenotypic characterization of circadian rhythms in stramenopiles and current efforts to determine the mechanisms of this 'brown clock'. The elucidation of this brown clock will enable a deeper understanding of the role of self-sustained oscillations in the adaptation to life in marine environments.

The Microalga Nannochloropsis during Transition from Quiescence to Autotrophy in Response to Nitrogen Availability.

The marine microalgae Nannochloropsis oceanica (CCMP1779) is a prolific producer of oil and is considered a viable and sustainable resource for biofuel feedstocks. Nitrogen (N) availability has a strong impact on the physiological status and metabolism of microalgal cells, but the exact nature of this response is poorly understood. To fill this gap we performed transcriptomic profiling combined with cellular and molecular analyses of N. oceanica CCMP1779 during the transition from quiescence to autotrophy. N deprivation-induced quiescence was accompanied by a strong reorganization of the photosynthetic apparatus and changes in the lipid homeostasis, leading to accumulation of triacylglycerol. Cell cycle activation and re-establishment of photosynthetic activity observed in response to resupply of the growth medium with N were accompanied by a rapid degradation of triacylglycerol stored in lipid droplets (LDs). Besides observing LD translocation into vacuoles, we also provide evidence for direct interaction between the LD surface protein (NoLDSP) and AUTOPHAGY-RELATED8 (NoATG8) protein and show a role of microlipophagy in LD turnover in N. oceanica CCMP1779. This knowledge is crucial not only for understanding the fundamental mechanisms controlling the cellular energy homeostasis in microalgal cells but also for development of efficient strategies to achieve higher algal biomass and better microalgal lipid productivity.

Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines.

Circadian clocks allow organisms to predict environmental changes caused by the rotation of the Earth. Although circadian rhythms are widespread among different taxa, the core components of circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have investigated cell division and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division occurred principally during the night period under diel conditions; however, these rhythms damped within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for the long-term monitoring of in vivo transcriptional rhythms in two Nannochlropsis species, Nannochloropsis oceanica CCMP1779 and Nannochloropsis salina CCMP537. The reporter lines express anticipatory behavior under light/dark cycles and free-running bioluminescence rhythms with periods of ~21-31 h that damped within ~3-4 days under constant light. Using different entrainment regimes, we demonstrate that these rhythms are modulated by a circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be modulated pharmacologically using a CK1 ε/δ inhibitor, suggesting a role of this kinase in the Nannochloropsis clock. Together with the molecular and genomic tools available for Nannochloropsis species, these reporter lines represent an excellent system for future studies on the molecular mechanisms of stramenopile circadian oscillators.

Fluctuating Light Interacts with Time of Day and Leaf Development Stage to Reprogram Gene Expression.

Natural light environments are highly variable. Flexible adjustment between light energy utilization and photoprotection is therefore of vital importance for plant performance and fitness in the field. Short-term reactions to changing light intensity are triggered inside chloroplasts and leaves within seconds to minutes, whereas long-term adjustments proceed over hours and days, integrating multiple signals. While the mechanisms of long-term acclimation to light intensity have been studied by changing constant growth light intensity during the day, responses to fluctuating growth light intensity have rarely been inspected in detail. We performed transcriptome profiling in Arabidopsis (Arabidopsis thaliana) leaves to investigate long-term gene expression responses to fluctuating light (FL). In particular, we examined whether responses differ between young and mature leaves or between morning and the end of the day. Our results highlight global reprogramming of gene expression under FL, including that of genes related to photoprotection, photosynthesis, and photorespiration and to pigment, prenylquinone, and vitamin metabolism. The FL-induced changes in gene expression varied between young and mature leaves at the same time point and between the same leaves in the morning and at the end of the day, indicating interactions of FL acclimation with leaf development stage and time of day. Only 46 genes were up- or down-regulated in both young and mature leaves at both time points. Combined analyses of gene coexpression and cis-elements pointed to a role of the circadian clock and light in coordinating the acclimatory responses of functionally related genes. Our results also suggest a possible cross talk between FL acclimation and systemic acquired resistance-like gene expression in young leaves.

Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779.

Utilization of microalgae has been hampered by limited tools for creating loss-of-function mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalgal lipid metabolism. We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal DNA after the removal of selection pressure, resulting in marker-free nontransgenic engineered lines. To test this system, we disrupted the nitrate reductase gene ( NR) and subsequently removed the CRISPR episome to generate nontransgenic marker-free nitrate reductase knockout lines (NR-KO).

Advanced genetic tools enable synthetic biology in the oleaginous microalgae Nannochloropsis sp.

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.

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production.

Nannochloropsis oceanica is an oleaginous microalga rich in ω3 long-chain polyunsaturated fatty acids (LC-PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC-PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC-PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779, we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC-OX and pNOC-stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase-encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC-PUFAs, with the overexpression of Δ12 and Δ5 FAD encoding sequences leading to an increase in the final ω3 product, EPA.

Genome diversity of tuber-bearing Solanum uncovers complex evolutionary history and targets of domestication in the cultivated potato.

Cultivated potatoes (Solanum tuberosum L.), domesticated from wild Solanum species native to the Andes of southern Peru, possess a diverse gene pool representing more than 100 tuber-bearing relatives (Solanum section Petota). A diversity panel of wild species, landraces, and cultivars was sequenced to assess genetic variation within tuber-bearing Solanum and the impact of domestication on genome diversity and identify key loci selected for cultivation in North and South America. Sequence diversity of diploid and tetraploid Stuberosum exceeded any crop resequencing study to date, in part due to expanded wild introgressions following polyploidy that captured alleles outside of their geographic origin. We identified 2,622 genes as under selection, with only 14-16% shared by North American and Andean cultivars, showing that a limited gene set drove early improvement of cultivated potato, while adaptation of upland (Stuberosum group Andigena) and lowland (S. tuberosum groups Chilotanum and Tuberosum) populations targeted distinct loci. Signatures of selection were uncovered in genes controlling carbohydrate metabolism, glycoalkaloid biosynthesis, the shikimate pathway, the cell cycle, and circadian rhythm. Reduced sexual fertility that accompanied the shift to asexual reproduction in cultivars was reflected by signatures of selection in genes regulating pollen development/gametogenesis. Exploration of haplotype diversity at potato's maturity locus (StCDF1) revealed introgression of truncated alleles from wild species, particularly Smicrodontum in long-day-adapted cultivars. This study uncovers a historic role of wild Solanum species in the diversification of long-day-adapted tetraploid potatoes, showing that extant natural populations represent an essential source of untapped adaptive potential.

Correction: Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779.

[This corrects the article DOI: 10.1371/journal.pgen.1003064.].

Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts.

BACKGROUND: Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail. RESULTS: Based on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds. CONCLUSIONS: Taken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.

A G-Box-Like Motif Is Necessary for Transcriptional Regulation by Circadian Pseudo-Response Regulators in Arabidopsis.

PSEUDO-RESPONSE REGULATORs (PRRs) play overlapping and distinct roles in maintaining circadian rhythms and regulating diverse biological processes, including the photoperiodic control of flowering, growth, and abiotic stress responses. PRRs act as transcriptional repressors and associate with chromatin via their conserved C-terminal CCT (CONSTANS, CONSTANS-like, and TIMING OF CAB EXPRESSION 1 [TOC1/PRR1]) domains by a still-poorly understood mechanism. Here, we identified genome-wide targets of PRR9 using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and compared them with PRR7, PRR5, and TOC1/PRR1 ChIP-seq data. We found that PRR binding sites are located within genomic regions of low nucleosome occupancy and high DNase I hypersensitivity. Moreover, conserved noncoding regions among Brassicaceae species are enriched around PRR binding sites, indicating that PRRs associate with functionally relevant cis-regulatory regions. The PRRs shared a significant number of binding regions, and our results indicate that they coordinately restrict the expression of target genes to around dawn. A G-box-like motif was overrepresented at PRR binding regions, and we showed that this motif is necessary for mediating transcriptional regulation of CIRCADIAN CLOCK ASSOCIATED 1 and PRR9 by the PRRs. Our results further our understanding of how PRRs target specific promoters and provide an extensive resource for studying circadian regulatory networks in plants.

Transcriptional coordination of physiological responses in Nannochloropsis oceanica CCMP1779 under light/dark cycles.

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.

The regulation of UV-B responses by the circadian clock.

The circadian clock modulates plant responses to environmental stimuli. In a recent study we showed that light and the circadian clock regulate daily changes in sensitivity to short treatments of high UV-B. Here we demonstrate that these time dependent changes in UV-B stress sensitivity are not mediated by the UV-B receptor UV resistantce locus 8. We also discuss the potential mechanisms involved in this process and the role of the circadian clock in the acclimation to UV-B.

Prevalence, evolution, and cis-regulation of diel transcription in Chlamydomonas reinhardtii.

Endogenous (circadian) and exogenous (e.g., diel) biological rhythms are a prominent feature of many living systems. In green algal species, knowledge of the extent of diel rhythmicity of genome-wide gene expression, its evolution, and its cis-regulatory mechanism is limited. In this study, we identified cyclically expressed genes under diel conditions in Chlamydomonas reinhardtii and found that ~50% of the 17,114 annotated genes exhibited cyclic expression. These cyclic expression patterns indicate a clear succession of biological processes during the course of a day. Among 237 functional categories enriched in cyclically expressed genes, >90% were phase-specific, including photosynthesis, cell division, and motility-related processes. By contrasting cyclic expression between C. reinhardtii and Arabidopsis thaliana putative orthologs, we found significant but weak conservation in cyclic gene expression patterns. On the other hand, within C. reinhardtii cyclic expression was preferentially maintained between duplicates, and the evolution of phase between paralogs is limited to relatively minor time shifts. Finally, to better understand the cis regulatory basis of diel expression, putative cis-regulatory elements were identified that could predict the expression phase of a subset of the cyclic transcriptome. Our findings demonstrate both the prevalence of cycling genes as well as the complex regulatory circuitry required to control cyclic expression in a green algal model, highlighting the need to consider diel expression in studying algal molecular networks and in future biotechnological applications.

Light and the circadian clock mediate time-specific changes in sensitivity to UV-B stress under light/dark cycles.

In Arabidopsis, the circadian clock regulates UV-B-mediated changes in gene expression. Here it is shown that circadian clock components are able to inhibit UV-B-induced gene expression in a gene-by-gene-specific manner and act downstream of the initial UV-B sensing by COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and UVR8 (UV RESISTANCE LOCUS 8). For example, the UV-B induction of ELIP1 (EARLY LIGHT INDUCIBLE PROTEIN 1) and PRR9 (PSEUDO-RESPONSE REGULATOR 9) is directly regulated by LUX (LUX ARRYTHMO), ELF4 (EARLY FLOWERING 4), and ELF3. Moreover, time-dependent changes in plant sensitivity to UV-B damage were observed. Wild-type Arabidopsis plants, but not circadian clock mutants, were more sensitive to UV-B treatment during the night periods than during the light periods under diel cycles. Experiments performed under short cycles of 6h light and 6h darkness showed that the increased stress sensitivity of plants to UV-B in the dark only occurred during the subjective night and not during the subjective day in wild-type seedlings. In contrast, the stress sensitivity of Arabidopsis mutants with a compromised circadian clock was still influenced by the light condition during the subjective day. Taken together, the results show that the clock and light modulate plant sensitivity to UV-B stress at different times of the day.

Effects of light and circadian clock on growth and chlorophyll accumulation of Nannochloropsis gaditana.

Circadian clocks synchronize various physiological, metabolic and developmental processes of organisms with specific phases of recurring changes in their environment (e.g. day and night or seasons). Here, we investigated whether the circadian clock plays a role in regulation of growth and chlorophyll (Chl) accumulation in Nannochloropsis gaditana, an oleaginous marine microalga which is considered as a potential feedstock for biofuels and for which a draft genome sequence has been published. Optical density (OD) of N. gaditana culture was monitored at 680 and 735 nm under 12:12 h or 18:6 h light-dark (LD) cycles and after switching to continuous illumination in photobioreactors. In parallel, Chl fluorescence was measured to assess the quantum yield of photosystem II. Furthermore, to test if red- or blue-light photoreceptors are involved in clock entrainment in N. gaditana, some of the experiments were conducted by using only red or blue light. Growth and Chl accumulation were confined to light periods in the LD cycles, increasing more strongly in the first half than in the second half of the light periods. After switching to continuous light, rhythmic oscillations continued (especially for OD680 ) at least in the first 24 h, with a 50% decrease in the capacity to grow and accumulate Chl during the first subjective night. Pronounced free-running oscillations were induced by blue light, but not by red light. In contrast, the photosystem II quantum yield was determined by light conditions. The results indicate interactions between circadian and light regulation of growth and Chl accumulation in N. gaditana.