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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.

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 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.

SummaryCircadian 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 inNannochloropsisspecies. In the four strains tested, cell division occurred principally during the night period under diel conditions, however, rhythms dampened within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for long-term monitoring ofin vivotranscriptional rhythms in twoNannochlropsisspecies,N. oceanicaCCMP1779 andN. salinaCCMP537. The reporter lines express free-running bioluminescence rhythms with periods of ~21-31 h that dampen within ~3-4 days under constant light. Using different entrainment regimes, we demonstrate that these rhythms are regulated 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 theNannochloropsisclock. Together with the molecular and genomic tools available forNannochloropsisspecies, these reporter lines represent an excellent system for future studies on the molecular mechanisms of stramenopile circadian oscillators.Significance statementStramenopiles are a large and diverse line of eukaryotes in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have developed bioluminescence reporter lines inNannochloropsisspecies and provide evidence for the presence of a circadian oscillator in stramenopiles; these lines will serve as tools for future studies to uncover the molecular mechanisms of circadian oscillations in these species.

Nannochloropsis oceanica, as other stramenopile microalgae, is rich in long-chain polyunsaturated fatty acids (LC-PUFA) such as eiconsapentaenoic acid (EPA). We observed that fatty acid desaturases (FAD) 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-sensing (LOV) domain that associates with a flavin mononucleotide and is able to sense blue light. These proteins also contain a bZIP DNA binding motif and can act as blue light regulated transcription factors by associating with a 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 any of the three aureochromes led to a decrease in the blue light specific induction of several FADs in CCMP1779. EPA content was also significantly reduced in NoAureo 2 and NoAureo 4 mutants. Taken together, our results indicate that aureochromes mediate blue light dependent regulation of LC-PUFA content in N. oceanica CCMP1779 cells.

International audience; 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.

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.

Nannochloropsis oceanica is an oleaginous microalga rich in ω3long-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. This article is protected by copyright. All rights reserved.

Huang et al. (1) argue that variant calling methods less conservative than GATK’s Best Practices workflow (2) increased false-positive variant discovery in our study of wild and cultivated potatoes (3), impacting diversity estimates. We disagree with their conclusion and highlight the rationale for the variant calling methods used in our study. First, GATK Best Practices, developed at the Broad Institute, were specifically designed and optimized for human genomics and medical research. Variant calling methodology for human medical research utilizes parameters and validation thresholds not intended for universal application across genomic studies. GATK’s hard filter is openly presented as a bias-prone substitute to their preferred (human-specific) variant quality … [↵][1]1To whom correspondence should be addressed. Email: buell{at}msu.edu. [1]: #xref-corresp-1-1

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.

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.

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.

Significance Worldwide, potato is the third most important crop grown for direct human consumption, but breeders have struggled to produce new varieties that outperform those released over a century ago, as evidenced by the most widely grown North American cultivar (Russet Burbank) released in 1876. Despite its importance, potato genetic diversity at the whole-genome level remains largely unexplored. Analysis of cultivated potato and its wild relatives using modern genomics approaches can provide insight into the genomic diversity of extant germplasm, reveal historic introgressions and hybridization events, and identify genes targeted during domestication that control variance for agricultural traits, all critical information to address food security in 21st century agriculture., 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 S. tuberosum 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 (S. tuberosum 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 S. microdontum 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.

Nannochloropsis oceanica CCMP1779 is an oleaginous heterokont microalga that is particularly amenable to synthetic biology approaches. The underlying complex genetic engineering often requires the expression of multiple transgenes (gene stacking). We integrated several techniques into a vector toolkit for multi-gene expression, including: multiple resistance marker genes, bidirectional promoters, and assembly of multiple expression cassettes into a single vector. We developed a series of gateway entry and destination vectors for assembly of two bidirectional promoter expression cassettes, which facilitates combinatorial high-capacity gene stacking. Several endogenous bidirectional promoters were demonstrated to be effective for driving transgene expression, with a dual-luciferase reporter system used to monitor transgene expression under different environmental conditions. The bidirectional promoter between the nitrate reductase and the nitrate transporter genes drives nitrogen source dependent transcription, enabling conditional transgene expression. Lethal concentrations of the antibiotics, Blasticidin, G418, and Nourseothricin, were determined and the corresponding marker genes used for genomic transformation selection.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0686-8) contains supplementary material, which is available to authorized users.

Circadian regulated changes in growth rates have been observed in numerous plants as well as in unicellular and multicellular algae. The circadian clock regulates a multitude of factors that affect growth in plants, such as water and carbon availability and light and hormone signalling pathways. The combination of high-resolution growth rate analyses with mutant and biochemical analysis is helping us elucidate the time-dependent interactions between these factors and discover the molecular mechanisms involved. At the molecular level, growth in plants is modulated through a complex regulatory network, in which the circadian clock acts at multiple levels.

In plants, the circadian clock functions as an endogenous pacemaker that anticipates and responds to a changing environment in order to optimize the timing of physiological and developmental events. Nusinow et al. elucidate the mechanism by which the circadian clock controls growth of the model plant Arabidopsis thaliana. A novel trimeric complex called the evening complex is regulated by the clock and has a peak of expression at dusk. The complex represses the expression of two transcription factors, PIF4 and PIF5, which are part of a light-signalling cascade that controls the timing of plant growth in response to light conditions. The circadian clock is required for adaptive responses to daily and seasonal changes in environmental conditions1,2,3. Light and the circadian clock interact to consolidate the phase of hypocotyl cell elongation to peak at dawn under diurnal cycles in Arabidopsis thaliana4,5,6,7. Here we identify a protein complex (called the evening complex)—composed of the proteins encoded by EARLY FLOWERING 3 (ELF3), ELF4 and the transcription-factor-encoding gene LUX ARRHYTHMO (LUX; also known as PHYTOCLOCK 1)—that directly regulates plant growth8,9,10,11,12. ELF3 is both necessary and sufficient to form a complex between ELF4 and LUX, and the complex is diurnally regulated, peaking at dusk. ELF3, ELF4 and LUX are required for the proper expression of the growth-promoting transcription factors encoded by PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 (also known as PHYTOCHROME INTERACTING FACTOR 3-LIKE 6) under diurnal conditions4,6,13. LUX targets the complex to the promoters of PIF4 and PIF5 in vivo. Mutations in PIF4 and/or PIF5 are epistatic to the loss of the ELF4–ELF3–LUX complex, suggesting that regulation of PIF4 and PIF5 is a crucial function of the complex. Therefore, the evening complex underlies the molecular basis for circadian gating of hypocotyl growth in the early evening.

The expression of a heterologous invertase in potato tubers (Solanum tuberosum) in either the cytosol or apoplast leads to a decrease in total sucrose content and to an increase in glucose. Depending on the targeting of the enzyme different changes in phenotype and metabolism of the tubers occur: the cytosolic invertase expressing tubers show an increase in the glycolytic flux, accumulation of amino acids and organic acids, and the appearance of novel disaccharides; however, these changes are not observed when the enzyme is expressed in the apoplast [Roessner et al. (2001). Plant Cell, 13, 11-29]. The analysis of these lines raised several questions concerning the regulation of compartmentation of metabolites in potato tubers. In the current study we addressed these questions by performing comparative subcellular metabolite profiling. We demonstrate that: (i) hexoses accumulate in the vacuole independently of their site of production, but that the cytosolic invertase expression led to a strong increase in the cytosolic glucose concentration and decrease in cytosolic sucrose, whereas these effects were more moderate in the apoplastic expressors; (ii) three out of four of the novel compounds found in the cytosolic overexpressors accumulate in the same compartment; (iii) despite changes in absolute cellular content the subcellular distribution of amino acids was invariant in the invertase overexpressing tubers. These results are discussed in the context of current models of the compartmentation of primary metabolism in heterotrophic plant tissues. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-008-0107-5) contains supplementary material, which is available to authorized users.

The pseudoresponse regulators (PRRs) participate in the progression of the circadian clock in Arabidopsis thaliana. The founding member of the family, TIMING OF CAB EXPRESSION1 (TOC1), is an essential component of the transcriptional network that constitutes the core mechanism of the circadian oscillator. Recent data suggest a role in circadian regulation for all five members of the PRR family; however, the molecular function of TOC1 or any other PRRs remains unknown. In this work, we present evidence for the involvement of PRR3 in the regulation of TOC1 protein stability. PRR3 was temporally coexpressed with TOC1 under different photoperiods, yet its tissue expression was only partially overlapping with that of TOC1, as PRR3 appeared restricted to the vasculature. Decreased expression of PRR3 resulted in reduced levels of TOC1 protein, while overexpression of PRR3 caused an increase in the levels of TOC1, all without affecting the amount of TOC1 transcript. PRR3 was able to bind to TOC1 in yeast and in plants and to perturb TOC1 interaction with ZEITLUPE (ZTL), which targets TOC1 for proteasome-dependent degradation. Together, our results indicate that PRR3 might function to modulate TOC1 stability by hindering ZTL-dependent TOC1 degradation, suggesting the existence of local regulators of clock activity and adding to the growing importance of posttranslational regulation in the design of circadian timing mechanisms in plants.

Interlocking transcriptional loops and regulated protein degradation are the principal mechanisms involved in the generation of self-sustaining circadian rhythms in many organisms. In Arabidopsis the first proposed regulatory transcriptional loop involved the transcription factors CIRCADIAN CLOCK ASSOCIATED (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the pseudo-response regulator TIMING OF CHLOROPHYLL A/B BINDING PROTEIN (TOC1/PRR1). Recent findings indicate that the TOC1 homologues PRR7 and PRR9 might also be involved in transcriptional regulatory loops with CCA1 and LHY. In this study we show that the overexpression of PRR7 in Arabidopsis leads to severely compromised circadian rhythms. These transgenic lines display significantly reduced levels of CCA1 and LHY RNA, providing further evidence for a transcriptional feedback loop between PRR7 and these transcription factors. In addition, we show that the PRR7 protein is phosphorylated in a circadian regulated manner and that its levels are post-translationally regulated by both diurnal and circadian mechanisms. The Arabidopsis circadian oscillator is therefore likely to be entrained to light/dark cycles both through transcriptional and post-transcriptional mechanisms.

Summary 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.

Transcriptional and allosteric regulation of ADP-Glc pyrophosphorylase (AGPase) plays a major role in the regulation of starch synthesis. Analysis of the response after detachment of growing potato tubers from the mother plant revealed that this concept requires extension. Starch synthesis was inhibited within 24 h of tuber detachment, even though the catalytic subunit of AGPase (AGPB) and overall AGPase activity remained high, the substrates ATP and Glc-1-P increased, and the glycerate-3-phosphate/inorganic orthophosphate (the allosteric activator and inhibitor, respectively) ratio increased. This inhibition was abolished in transformants in which a bacterial AGPase replaced the potato AGPase. Measurements of the subcellular levels of each metabolite between Suc and starch established AGPase as the only step whose substrates increase and mass action ratio decreases after detachment of wild-type tubers. Separation of extracts on nonreducing SDS gels revealed that AGPB is present as a mixture of monomers and dimers in growing tubers and becomes dimerized completely in detached tubers. Dimerization led to inactivation of the enzyme as a result of a marked decrease of the substrate affinity and sensitivity to allosteric effectors. Dimerization could be reversed and AGPase reactivated in vitro by incubating extracts with DTT. Incubation of tuber slices with DTT or high Suc levels reduced dimerization, increased AGPase activation, and stimulated starch synthesis in vivo. In intact tubers, the Suc content correlated strongly with AGPase activation across a range of treatments, including tuber detachment, aging of the mother plant, heterologous overexpression of Suc phosphorylase, and antisense inhibition of endogenous AGPase activity. Furthermore, activation of AGPase resulted in a stimulation of starch synthesis and decreased levels of glycolytic intermediates.

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

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.

The compartmentation of metabolism in heterotrophic plant tissues is poorly understood due to the lack of data on metabolite distributions and fluxes between subcellular organelles. The main reason for this is the lack of suitable experimental methods with which intracellular metabolism can be measured. Here, we describe a nonaqueous fractionation method that allows the subcellular distributions of metabolites in developing potato (Solanum tuberosum L. cv Desiree) tubers to be calculated. In addition, we have coupled this fractionation method to a recently described gas chromatography-mass spectrometry procedure that allows the measurement of a wide range of small metabolites. To calculate the subcellular metabolite concentrations, we have analyzed organelle volumes in growing potato tubers using electron microscopy. The relative volume distributions in tubers are very similar to the ones for source leaves. More than 60% of most sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrations of these metabolites is often higher in the cytosol. Significant amounts of the substrates for starch biosynthesis, hexose phosphates, and ATP were found in the plastid. However, pyrophosphate was located almost exclusively in the cytosol. Calculation of the mass action ratios of sucrose synthase, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucomutase indicate that these enzymes are close to equilibrium in developing potato tubers. However, due to the low plastidic pyrophosphate concentration, the reaction catalyzed by ADP-glucose pyrophosphorylase was estimated to be far removed from equilibrium.

Potato is a globally important crop. Unfortunately, potato farming is plagued with problems associated with the sprouting behavior of seed tubers. The data presented here demonstrate that using transgenic technology can influence this behavior. Transgenic tubers cytosolically expressing an inorganic pyrophosphatase gene derived from Escherichia coli under the control of the tuber-specific patatin promoter display significantly accelerated sprouting. The period of presprouting dormancy for transgenic tubers planted immediately after harvest is reduced by six to seven weeks when compared to wild-type tubers. This study demonstrates a method with which to regulate dormancy, an important aspect of potato crop management.

The early stages of tuber development are characterized by cell division, high metabolic activity, and the predominance of invertase as the sucrose (Suc) cleaving activity. However, during the subsequent phase of starch accumulation the cleavage of Suc occurs primarily by the action of Suc synthase. The mechanism that is responsible for this switch in Suc cleaving activities is currently unknown. One striking difference between the invertase and Suc synthase mediated cleavage of Suc is the direct involvement of inorganic pyrophosphate (PPi) in the latter case. There is presently no convincing explanation of how the PPi required to support this process is generated in potato (Solanum tuberosum) tubers. The major site of PPi production in a maturing potato tubers is likely to be the reaction catalyzed by ADP-glucose pyrophosphorylase, the first committed step of starch biosynthesis in amyloplasts. We present data based on the analysis of the PPi levels in various transgenic plants altered in starch and Suc metabolism that support the hypothesis that PPi produced in the plastid is used to support cytosolic Suc breakdown and that PPi is an important coordinator of cytosolic and plastidial metabolism in potato tubers.

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., Author Summary Algae are a highly diverse group of organisms that have become the focus of renewed interest due to their potential for producing biofuel feedstocks, nutraceuticals, and biomaterials. Their high photosynthetic yields and ability to grow in areas unsuitable for agriculture provide a potential sustainable alternative to using traditional agricultural crops for biofuels. Because none of the algae currently in use have a history of domestication, and bioengineering of algae is still in its infancy, there is a need to develop algal strains adapted to cultivation for industrial large-scale production of desired compounds. Model organisms ranging from mice to baker's yeast have been instrumental in providing insights into fundamental biological structures and functions. The algal field needs versatile models to develop a fundamental understanding of photosynthetic production of biomass and valuable compounds in unicellular, marine, oleaginous algal species. To contribute to the development of such an algal model system for basic discovery, we sequenced the genome and two sets of transcriptomes of N. oceanica CCMP1779, assembled the genomic sequence, identified putative genes, and began to interpret the function of selected genes. This species was chosen because it is readily transformable with foreign DNA and grows well in culture.

Up to 30% of the plant transcriptome is circadian clock-regulated in different species; however, we still lack a good understanding of the mechanisms involved in these genome-wide oscillations in gene expression. Here, we show that PSEUDO-RESPONSE REGULATOR 7 (PRR7), a central component of the Arabidopsis clock, is directly involved in the repression of master regulators of plant growth, light signaling and stress responses. The expression levels of most PRR7 target genes peak around dawn, in an antiphasic manner to PRR7 protein levels, and were repressed by PRR7. These findings indicate that PRR7 is important for cyclic gene expression by repressing the transcription of morning-expressed genes. In particular we found an enrichment of the genes involved in abiotic stress responses, and in accordance we observed that PRR7 is involved in the oxidative stress response and the regulation of stomata conductance.

Primary metabolism in plants is tightly regulated by environmental factors such as light and nutrient availability at multiple levels. The circadian clock is a self-sustained endogenous oscillator that enables organisms to predict daily and seasonal changes. The regulation of primary metabolism by the circadian clock has been proposed to explain the importance of circadian rhythms in plant growth and survival. Recent transcriptomic and metabolomic analyses indicate a wide spread circadian regulation of different metabolic processes. We review evidence of circadian regulation of pathways in primary metabolism, discuss the challenges faced for discerning the mechanisms regulating circadian metabolic oscillations and present recent evidence of regulation of the circadian clock by metabolites.

The C-REPEAT BINDING FACTOR (CBF) cold-response pathway has a prominent role in cold acclimation, the process whereby certain plants increase tolerance to freezing in response to low nonfreezing temperatures. In Arabidopsis , the CBF pathway is characterized by rapid induction of the C-REPEAT BINDING FACTOR 1 ( CBF1 ), CBF2 , and CBF3 genes, which encode transcriptional activators, followed by induction of the CBF-targeted genes known as the “CBF regulon.” Expression of the CBF regulon results in an increase in freezing tolerance. Previous studies established that CBF1, CBF2 , and CBF3 are subject to circadian regulation and that their cold induction is gated by the circadian clock. Here we present the results of genetic analysis and ChIP experiments indicating that both these forms of regulation involve direct positive action of two transcription factors that are core components of the clock, i.e., CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). In plants carrying the cca1 -11/ lhy -21 double mutation, cold induction of CBF1 , CBF2 , and CBF3 was greatly impaired, and circadian regulation of CBF1 and CBF3 was essentially eliminated; circadian regulation of CBF2 continued, although with significantly reduced amplitude. Circadian regulation and cold induction of three CBF regulon genes, i.e., COLD-REGULATED GENE15A ( COR15A ), COR47 , and COR78 , also were greatly diminished in plants carrying the cca1 -11/ lhy -21 double mutation. Furthermore, the cca1 -11/ lhy -21 double mutation resulted in impaired freezing tolerance in both nonacclimated and cold-acclimated plants. These results indicate that CCA1/LHY-mediated output from the circadian clock contributes to plant cold tolerance through regulation of the CBF cold-response pathway.

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.

The core mechanism of the circadian oscillators described to date rely on transcriptional negative feedback loops with a delay between the negative and the positive components . In plants, the first suggested regulatory loop involves the transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the pseudo-response regulator TIMING OF CAB EXPRESSION 1 (TOC1/PRR1). TOC1 is a member of the Arabidopsis circadian-regulated PRR gene family . Analysis of single and double mutants in PRR7 and PRR9 indicates that these morning-expressed genes play a dual role in the circadian clock, being involved in the transmission of light signals to the clock and in the regulation of the central oscillator. Furthermore, CCA1 and LHY had a positive effect on PRR7 and PRR9 expression levels, indicating that they might form part of an additional regulatory feedback loop. We propose that the Arabidopsis circadian oscillator is composed of several interlocking positive and negative feedback loops, a feature of clock regulation that appears broadly conserved between plants, fungi, and animals. Fil: Farré, Eva M.. The Scripps Research Institute; Estados Unidos Fil: Harmer, Stacey L.. University of California; Estados Unidos Fil: Harmon, Frank G.. The Scripps Research Institute; Estados Unidos Fil: Yanovsky, Marcelo Javier. The Scripps Research Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Kay, Steve A.. The Scripps Research Institute; Estados Unidos

We developed a mathematical model of the Arabidopsis circadian clock, including PRR7 and PRR9, which is able to predict several single, double and triple mutant phenotypes. Sensitivity Analysis was used to identify the properties and time sensing mechanisms of model structures. PRR7 and CCA1/LHY were identified as weak points of the mathematical model indicating where more experimental data is needed for further model development. Detailed dynamical studies showed that the timing of an evening light sensing element is essential for day length responsiveness, In recent years, molecular genetic techniques have revealed a complex network of components in the Arabidopsis circadian clock. Mathematical models allow for a detailed study of the dynamics and architecture of such complex gene networks leading to a better understanding of the genetic interactions. It is important to maintain a constant iteration with experimentation, to include novel components as they are discovered and use the updated model to design new experiments. This study develops a framework to introduce new components into the mathematical model of the Arabidopsis circadian clock accelerating the iterative model development process and gaining insight into the system's properties. We used the interlocked feedback loop model published in Locke et al (2005) as the base model. In Arabidopsis, the first suggested regulatory loop involves the morning expressed transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and the evening expressed pseudo-response regulator TIMING OF CAB EXPRESSION (TOC1). The hypothetical component X had been introduced to realize a longer delay between gene expression of CCA1/LHY and TOC1. The introduction of Y was motivated by the need for a mechanism to reproduce the dampening short period rhythms of the cca1/lhy double mutant and to include an additional light input at the end of the day. In this study, the new components pseudo-response regulators PRR7 and PRR9 were added in negative feedback loops based on the biological hypothesis that they are activated by LHY and in turn repress LHY transcription (Farré et al, 2005; Figure 1). We present three iterations steps of model development (Figure 1A–C). A wide range of tools was used to establish and analyze new model structures. One of the challenges facing mathematical modeling of biological processes is parameter identification; they are notoriously difficult to determine experimentally. We established an optimization procedure based on an evolutionary strategy with a cost function mainly derived from wild-type characteristics. This ensured that the model was not restricted by a specific set of parameters and enabled us to use a large set of biological mutant information to assess the predictive capability of the model structure. Models were evaluated by means of an extended phenotype catalogue, allowing for an easy and fair comparison of the structures. We also carried out detailed simulation analysis of component interactions to identify weak points in the structure and suggest further modifications. Finally, we applied sensitivity analysis in a novel manner, using it to direct the model development. Sensitivity analysis provides quantitative measures of robustness; the two measures in this study were the traces of component concentrations over time (classical state sensitivities) and phase behavior (measured by the phase response curve). Three major results emerged from the model development process. First, the iteration process helped us to learn about general characteristics of the system. We observed that the timing of Y expression is critical for evening light entrainment, which enables the system to respond to changes in day length. This is important for our understanding of the mechanism of light input to the clock and will add in the identification of biological candidates for this function. In addition, our results suggest that a detailed description of the mechanisms of genetic interactions is important for the systems behavior. We observed that the introduction of an experimentally based precise light regulation mechanism on PRR9 expression had a significant effect on the systems behavior. Second, the final model structure (Figure 1C) was capable of predicting a wide range of mutant phenotypes, such as a reduction of TOC1 expression by RNAi (toc1RNAi), mutations in PRR7 and PRR9 and the novel mutant combinations prr9toc1RNAi and prr7prr9toc1RNAi. However, it was unable to predict the mutations in CCA1 and LHY. Finally, sensitivity analysis identified the weak points of the system. The developed model structure was heavily based on the TOC1/Y feedback loop. This could explain the model's failure to predict the cca1lhy double mutant phenotype. More detailed information on the regulation of CCA1 and LHY expression will be important to achieve the right balance between the different regulatory loops in the mathematical model. This is in accordance with genetic studies that have identified several genes involved in the regulation of LHY and CCA1 expression. The identification of their mechanism of action will be necessary for the next model development., In plants, as in animals, the core mechanism to retain rhythmic gene expression relies on the interaction of multiple feedback loops. In recent years, molecular genetic techniques have revealed a complex network of clock components in Arabidopsis. To gain insight into the dynamics of these interactions, new components need to be integrated into the mathematical model of the plant clock. Our approach accelerates the iterative process of model identification, to incorporate new components, and to systematically test different proposed structural hypotheses. Recent studies indicate that the pseudo-response regulators PRR7 and PRR9 play a key role in the core clock of Arabidopsis. We incorporate PRR7 and PRR9 into an existing model involving the transcription factors TIMING OF CAB (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED (CCA1). We propose candidate models based on experimental hypotheses and identify the computational models with the application of an optimization routine. Validation is accomplished through systematic analysis of various mutant phenotypes. We introduce and apply sensitivity analysis as a novel tool for analyzing and distinguishing the characteristics of proposed architectures, which also allows for further validation of the hypothesized structures.