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Sophorolipids are biobased and biodegradable glycolipid surface-active agents contributing to the shift from petroleum to biobased surfactants, associated with clear environmental benefits. However, their production cost is currently too high to allow commercialisation. Therefore, a continuous sophorolipid production process was evaluated, i.e., a retentostat with an external filtration unit. Despite an initial increase in volumetric productivity, productivity eventually declined to almost 0gL-1 h-1. Following comprehensive metabolomics on supernatant obtained from a standardised retentostat, we hypothesised exhaustion of the N-starvation-induced autophagy as the main mechanism responsible for the decline in bolaform sophorolipid productivity. Thirty-six metabolites that correlate with RNA/protein autophagy and high sophorolipid productivity were putatively identified. In conclusion, our results unveil a plausible cause of this bola sophorolipid productivity decline in an industrially relevant bioreactor set-up, which may thus impact majorly on future yeast biosurfactant regulation studies and the finetuning of bola sophorolipid production processes.

Plants need to cope with strong variations of nitrogen availability in the soil. Although many molecular players are being discovered concerning how plants perceive NO(3)(−) provision, it is less clear how plants recognize a lack of nitrogen. Following nitrogen removal, plants activate their nitrogen starvation response (NSR), which is characterized by the activation of very high-affinity nitrate transport systems (NRT2.4 and NRT2.5) and other sentinel genes involved in N remobilization such as GDH3. Using a combination of functional genomics via transcription factor perturbation and molecular physiology studies, we show that the transcription factors belonging to the HHO subfamily are important regulators of NSR through two potential mechanisms. First, HHOs directly repress the high-affinity nitrate transporters, NRT2.4 and NRT2.5. hho mutants display increased high-affinity nitrate transport activity, opening up promising perspectives for biotechnological applications. Second, we show that reactive oxygen species (ROS) are important to control NSR in wild-type plants and that HRS1 and HHO1 overexpressors and mutants are affected in their ROS content, defining a potential feed-forward branch of the signaling pathway. Taken together, our results define the relationships of two types of molecular players controlling the NSR, namely ROS and the HHO transcription factors. This work (i) up opens perspectives on a poorly understood nutrient-related signaling pathway and (ii) defines targets for molecular breeding of plants with enhanced NO(3)(−) uptake.

Nannochloropsis oceanica F&M-M24 is able to increase its lipid content during nitrogen starvation to more than 50% of the total biomass. We investigated the ultrastructural changes and the variation in the content of main cell biomolecules that accompany the final phase of lipid accumulation. Nitrogen starvation induced a first phase of thylakoid disruption followed by chloroplast macroautophagy and formation of lipid droplets. During this phase, the total amount of proteins decreased by one-third, while carbohydrates decreased by 12–13%, suggesting that lipid droplets were formed by remodelling of chloroplast membranes and synthesis of fatty acids from carbohydrates and amino acids. The change in mitochondrial ultrastructure suggests also that these organelles were involved in the process. The cell wall increased its thickness and changed its structure during starvation, indicating that a disruption process could be partially affected by the increase in wall thickness for biomolecules recovery from starved cells. The wall thickness in strain F&M-M24 was much lower than that observed in other strains of N. oceanica, showing a possible advantage of this strain for the purpose of biomolecules extraction. The modifications following starvation were interpreted as a response to reduction of availability of a key nutrient (nitrogen). The result is a prolonged survival in quiescence until an improvement of the environmental conditions (nutrient availability) allows the rebuilding of the photosynthetic apparatus and the full recovery of cell functions.

The crystal structures of adenylylated and unadenylylated GlnK, a PII protein from Corynebacterium glutamicum, indicate that adenylylation of Tyr51 in the T-loop does not interfere with the PII-typical conformational changes that occur in the T-loop upon effector binding. Rather, T-loop adenylylation further expands the repertoire of mechanisms that enable PII function., PII proteins are ubiquitous signaling proteins that are involved in the regulation of the nitrogen/carbon balance in bacteria, archaea, and some plants and algae. Signal transduction via PII proteins is modulated by effector molecules and post-translational modifications in the PII T-loop. Whereas the binding of ADP, ATP and the concomitant binding of ATP and 2-oxoglutarate (2OG) engender two distinct conformations of the T-loop that either favor or disfavor the interaction with partner proteins, the structural consequences of post-translational modifications such as phosphorylation, uridylylation and adenylylation are far less well understood. In the present study, crystal structures of the PII protein GlnK from Corynebacterium glutamicum have been determined, namely of adenylylated GlnK (adGlnK) and unmodified unadenylylated GlnK (unGlnK). AdGlnK has been proposed to act as an inducer of the transcription repressor AmtR, and the adenylylation of Tyr51 in GlnK has been proposed to be a prerequisite for this function. The structures of unGlnK and adGlnK allow the first atomic insights into the structural implications of the covalent attachment of an AMP moiety to the T-loop. The overall GlnK fold remains unaltered upon adenylylation, and T-loop adenylylation does not appear to interfere with the formation of the two major functionally important T-loop conformations, namely the extended T-loop in the canonical ADP-bound state and the compacted T-loop that is adopted upon the simultaneous binding of Mg-ATP and 2OG. Thus, the PII-typical conformational switching mechanism appears to be preserved in GlnK from C. glutamicum, while at the same time the functional repertoire becomes expanded through the accommodation of a peculiar post-translational modification.

This scoping review is aimed at the application of the metabolomics platform to dissect key metabolites and their intermediates to observe the regulatory mechanisms of starvation-induced autophagy in Saccharomyces cerevisiae. Four research papers were shortlisted in this review following the inclusion and exclusion criteria. We observed a commonly shared pathway undertaken by S. cerevisiae under nutritional stress. Targeted and untargeted metabolomics was applied in either of these studies using varying platforms resulting in the annotation of several different observable metabolites. We saw a commonly shared pathway undertaken by S. cerevisiae under nutritional stress. Following nitrogen starvation, the concentration of cellular nucleosides was altered as a result of autophagic RNA degradation. Additionally, it is also found that autophagy replenishes amino acid pools to sustain macromolecule synthesis. Furthermore, in glucose starvation, nucleosides were broken down into carbonaceous metabolites that are being funneled into the non-oxidative pentose phosphate pathway. The ribose salvage allows for the survival of starved yeast. Moreover, acute glucose starvation showed autophagy to be involved in maintaining ATP/energy levels. We highlighted the practicality of metabolomics as a tool to better understand the underlying mechanisms involved to maintain homeostasis by recycling degradative products to ensure the survival of S. cerevisiae under starvation. The application of metabolomics has extended the scope of autophagy and provided newer intervention targets against cancer as well as neurodegenerative diseases in which autophagy is implicated.

Target of rapamycin (TOR) kinases form two distinct complexes, TORC1 and TORC2, which are evolutionarily conserved among eukaryotes. These complexes control intracellular biochemical processes in response to changes in extracellular nutrient conditions. Previous studies using the fission yeast, Schizosaccharomyces pombe, showed that the TORC2 signaling pathway, which is essential for cell proliferation under glucose-limited conditions, ensures cell-surface localization of a high-affinity hexose transporter, Ght5, by downregulating its endocytosis. The TORC2 signaling pathway retains Ght5 on the cell surface, depending on the presence of nitrogen sources in medium. Ght5 is transported to vacuoles upon nitrogen starvation. In this review, we discuss the molecular mechanisms underlying this regulation to cope with nutritional stress, a response which may be conserved from yeasts to mammals.

Both low temperature and nitrogen starvation caused chlorosis of cyanobacteria. Here, in this study, for the first time, we compared the effects of low temperature, nitrogen starvation, and their combination on the photosynthesis and metabolites of a thermophilic cyanobacterium strain, Thermosynechococcus E542. Under various culture conditions, the growth rates, pigment contents, and chlorophyll fluorescence were monitored, and the composition of alkanes, lipidomes, and carbohydrates were determined. It was found that low temperature (35 °C) significantly suppressed the growth of Thermosynechococcus E542. Nitrogen starvation at 45 °C and 55 °C did not affect the growth, however, combined treatment of low temperature and nitrogen starvation led to the lowest growth rate and biomass productivity. Both low temperature and nitrogen starvation caused significantly declined contents of pigments, but they resulted in a different effect on the OJIP curves, and their combination led to the lowest pigment contents. The composition of fatty acids and alkanes was altered upon low-temperature cultivation, while nitrogen starvation caused reduced contents of all lipids. The low temperature did not affect carbohydrate contents, while nitrogen starvation greatly enhanced carbohydrate content, and their combination did not enhance carbohydrate content, but led to reduced productivity. These results revealed the influence of low temperature, nitrogen starvation, and their combined treatment for the accumulation of phycobiliproteins, lipids, and carbohydrates of a thermophilic cyanobacterium strain, Thermosynechococcus E542.

The amount of intracellular trehalose increases in response to environmental stress in yeast (Saccharomyces cerevisiae). When that stress is terminated, the accumulated trehalose rapidly degrades into glucose rapidly. Synthesis of trehalose is fulfilled by the Trehalose Phosphate Synthase (TPS) enzyme complex, whereas the degradation of trehalose is done by the neutral trehalase enzyme. Under different stress conditions, transcription of the NTH1 gene is activated and Stress Response Elements (STRE) are required for this activation. Nrg1 protein can bind promoters including STRE and PDS elements. Because of the presence of three possible Nrg1 repressor binding sites on the NTH1 promoter, the NTH1 gene may be regulated by the Nrg1 repressor. In order to test this hypothesis, Δnrg1 mutant yeast and its isogenic wildtype yeast strain were used to analyze the transcriptional activation of the NTH1 gene under nitrogen starving conditions. Nth1 transcription of the mutant yeast was seven-fold higher than that of the wild-type under growth conditions, and was not changed during nitrogen starvation. The protein-DNA docking analysis also supported the possibility of Nrg1 binding to the NTH1 promoter. These results revealed that NTH1 gene expression is constitutive in the absence of the Nrg1 repressor protein, hence the transcription of NTH1 is repressed by the Nrg1 protein.

Sophora japonica is a native leguminous tree species in China. The high stress tolerance contributes to its long lifespan of thousands of years. The lack of genomic resources greatly limits genetic studies on the stress responses of S. japonica. In this study, RNA-seq was conducted for S. japonica roots grown under short-term 20% polyethylene glycol (PEG) 6000-induced drought stress under normal N and N starvation conditions (1 and 0 mM NH4NO3, respectively). In each of the libraries, we generated more than 25 million clean reads, which were then de novo assembled to 46,852 unigenes with an average length of 1310.49 bp. In the differential expression analyses, more differentially expressed genes (DEGs) were found under drought with N starvation than under single stresses. The number of transcripts identified under N starvation and drought in S. japonica was nearly the same, but more upregulated genes were induced by drought, while more downregulated genes were induced by N starvation. Genes involved in “phenylpropanoid biosynthesis” and “biosynthesis of amino acids” pathways were upregulated according to KEGG enrichment analyses, irrespective of the stress treatments. Additionally, upregulated N metabolism genes were enriched upon drought, and downregulated photosynthesis genes were enriched under N starvation. We found 4,372 and 5,430 drought-responsive DEGs under normal N and N starvation conditions, respectively. N starvation may aggravate drought by downregulating transcripts in the “carbon metabolism”, “ribosome”, “arginine biosynthesis pathway”, “oxidative phosphorylation” and “aminoacyl-tRNA biosynthesis” pathways. We identified 78 genes related to N uptake and assimilation, 38 of which exhibited differential expression under stress. A total of 395 DEGs were categorized as transcription factors, of which AR2/ERF-ERF, WRKY, NAC, MYB, bHLH, C3H and C2C2-Dof families played key roles in drought and N starvation stresses. The transcriptome data obtained, and the genes identified facilitate our understanding of the mechanisms of S. japonica responses to drought and N starvation stresses and provide a molecular foundation for understanding the mechanisms of its long lifespan for breeding resistant varieties for greening.

Significance In this work, we identified the regulatory mechanism of the key control point of cyanobacterial carbon metabolism, the glycolytic phosphoglycerate mutase (PGAM) reaction, converting 3-PGA into 2-PGA and thereby exporting organic carbon from the photosynthetic Calvin cycle. We show that PGAM activity is controlled by a small modulator protein PirC (product of sll0944), which inhibits the enzyme through protein–protein interaction. The availability of PirC for PGAM inhibition is controlled by the pervasive carbon/nitrogen balance regulator PII, which sequesters PirC at low 2-oxoglutarate levels and releases it at high 2-oxoglutarate levels. PirC-mediated inhibition of PGAM triggers glycogen accumulation, and disrupting this regulation allows the redirection of carbon flux, a decisive requirement for transforming cyanobacteria into green factories., Nitrogen limitation imposes a major transition in the lifestyle of nondiazotrophic cyanobacteria that is controlled by a complex interplay of regulatory factors involving the pervasive signal processor PII. Immediately upon nitrogen limitation, newly fixed carbon is redirected toward glycogen synthesis. How the metabolic switch for diverting fixed carbon toward the synthesis of glycogen or of cellular building blocks is operated was so far poorly understood. Here, using the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803 as model system, we identified a novel PII interactor, the product of the sll0944 gene, which we named PirC. We show that PirC binds to and inhibits the activity of 2,3-phosphoglycerate–independent phosphoglycerate mutase (PGAM), the enzyme that deviates newly fixed CO2 toward lower glycolysis. The binding of PirC to either PII or PGAM is tuned by the metabolite 2-oxoglutarate (2-OG), which accumulates upon nitrogen starvation. In these conditions, the high levels of 2-OG dissociate the PirC–PII complex to promote PirC binding to and inhibition of PGAM. Accordingly, a PirC-deficient mutant showed strongly reduced glycogen levels upon nitrogen deprivation, whereas polyhydroxybutyrate granules were overaccumulated compared to wild-type. Metabolome analysis revealed an imbalance in 3-phosphoglycerate to pyruvate levels in the pirC mutant, confirming that PirC controls the carbon flux in cyanobacteria via mutually exclusive interaction with either PII or PGAM.

Significance During genome analysis, genes encoding small proteins are frequently neglected. Accordingly, small proteins have remained underinvestigated in all domains of life. Based on a previous systematic search for such genes, we present the functional analysis of the 66 amino acids protein NblD in a photosynthetic cyanobacterium. We show that NblD plays a crucial role during the coordinated dismantling of phycobilisome light-harvesting complexes. This disassembly is triggered when the cells become starved for nitrogen, a condition that frequently occurs in nature. Similar to NblA that tags phycobiliproteins for proteolysis, NblD binds to phycocyanin polypeptides but has a different function. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration., Phycobilisomes are the major pigment–protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte, and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded following an intricate genetic program. Here, we describe the role of NblD, a cysteine-rich, small protein in this process in cyanobacteria. Deletion of the nblD gene in the cyanobacterium Synechocystis sp. PCC 6803 prevented the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype, which could be complemented by a plasmid-localized gene copy. Competitive growth experiments between the ΔnblD and the wild-type strain provided direct evidence for the physiological importance of NblD under nitrogen-limited conditions. Ectopic expression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation. Transcriptome analysis indicated increased nblA1/2 transcript levels in the ΔnblD strain during nitrogen starvation, implying that NblD does not act as a transcriptional (co)regulator. However, immunoprecipitation and far-western experiments identified the chromophorylated (holo form) of the phycocyanin β-subunit (CpcB) as its target, while apo-CpcB was not bound. The addition of recombinant NblD to isolated phycobilisomes caused a reduction in phycocyanin absorbance and a broadening and shifting of the peak to lower wavelengths, indicating the occurrence of structural changes. These data demonstrate that NblD plays a crucial role in the coordinated dismantling of phycobilisomes and add it as a factor to the genetically programmed response to nitrogen starvation.

Microalgae can accumulate substantial amounts of triacylglycerol, the primary feedstock for biodiesel production, under different stress conditions. In this study, exposure to alkaline pH (pH 10) using carbonate-bicarbonate buffer, nitrogen starvation, and both were applied to induce triacylglycerol accumulation in Auxenochlorella protothecoides KP7. In addition to triacylglycerol accumulation, growth, biochemical composition, and fatty acid profiles were examined in detail and compared. Our results illustrated that the combination of nitrogen starvation and alkaline pH led to significant increases in triacylglycerol and starch contents and drastic decreases in chlorophyll and protein contents compared with their control levels. Triacylglycerol levels, the triacylglycerol/total lipid ratio, and lipid productivities were significantly increased after 7 days of cultivation under the combined stress compared with the effects of nitrogen starvation alone. After prolonged cultivation under the combined stress, triacylglycerol content reached up to 25.01% of the dry cell weight and constituted up to 81.25% of total lipids. Under the combined stress, the major fatty acids in the total lipid of A. protothecoides KP7 were C18:1 (57.5%), C18:2 (19.5%), C16:0 (12.5%), and C18:3 (5.6%), and the percentages of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids represented 16.3, 58.4, and 25.1% of the total fatty acids, respectively. Furthermore, the combined stress was associated with the lowest iodine value (97.64 g I-2 (100 g)(-1)), highest oxidative stability (7.29 h), and highest cetane number (55.26) among the examined conditions. The use of nitrogen-free medium containing carbonate-bicarbonate buffer for cultivation appears promising for enhancing triacylglycerol production in A. protothecoides.

Microalgae are considered feedstock for biodiesel production due to their capability to accumulate triacylglycerols, which have a 99% conversion rate into biodiesel, under certain conditions. This study aims to evaluate thirty native microalgal strains as feedstock for biodiesel production based on their biomass and lipid productivities, and total lipid and triacylglycerol contents under nitrogen-sufcient and nitrogen starvation conditions. In addition, Chlamydomonas reinhardtii cw15 mutant strain was utilized as a reference strain for triacylglycerol accumulation. Among the eight potent strains, Chlorella vulgaris KP2 was considered as a most promising strain with the highest triacylglycerol content, highest total lipid content (28.56% of dry cell weight), and the highest lipid productivity (4.56 mg/L/day) under nitrogen starvation. Under nitrogen starvation, the major fatty acids in the triacylglycerol of Chlorella vulgaris KP2 were C18:1 (37.56%), C16:0 (23.16%), C18:0 (23.07), C18:2 (7.00%), and C18:3 (3.12%), and the percentages of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids represented 49.26, 38.73, and 10.12% of the total fatty acids, respectively. Furthermore, the fatty acid methyl esters of triacylglycerol displayed remarkable biodiesel properties with a lower iodine value (59.00 gI2/100 g), higher oxidative stability (14.24 h) and higher cetane number (58.73) under nitrogen starvation. This study suggests that nitrogenstarved Chlorella vulgaris KP2 could be used as a feedstock for biodiesel production due to the considerable amounts of triacylglycerol and favorable biodiesel properties. TÜBİTAK (The Scientific and Technological Research Council of Turkey, 116Y345) and the University of Nevsehir Hacı Bektas Veli, Scientifc Research Projects Unit (NEUBAP15F26)

Many microorganisms can accumulate biomass in the form of lipids and polysaccharides, which can be used for biofuels, bioplastics, food and feed. Some innovative bioprocesses exploit the competitive advantage provided by such accumulation ability, mainly under N-starvation, to select high-accumulating strains against biological contaminants, by using uncoupled nutrient feeding. However, there is no general and easily comparable parameter available to compare biomass accumulation ability among different microbial strains, which could measure the competitive advantage. Here, a parameter termed “fattening factor” (ηx) is described to quantify such strain-specific biomass accumulation ability in bacteria, yeasts and microalgae. This parameter measures how many fold a microbial population can increase its biomass just as the result of accumulation. It is derived from considerations about the main metabolic aspects of cells’ response to N-starvation, which induces variations in cell cycle, biomass production and biochemical composition. The fattening factor described here should be easily estimatable in N-starvation for every culturable microbial strain, by measuring the amount of accumulated biomass.

The microalgae Rhodomonas sp. is known as an excellent feed source for live feed organisms such as copepods. The main benefits of feeding Rhodomonas to live feed animals are attributed to the relative high polyunsaturated fatty acid (PUFA) level, the combination of containing both docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and the ratio between these fatty acids (FA). It has been shown that microalgae are able to accumulate valuable metabolites, such as lipids, under adverse conditions. The easiest and most inexpensive method to induce stress to microalgae is through nitrogen (N) starvation. In this study, the effect of N-starvation on biomass concentration, cell volume, and cellular composition, such as fatty acid concentration and composition, and phycoerythrin (PE) concentration of Rhodomonas sp. during a period of 8 days, was investigated. The research was divided into two stages. In the first (growth) stage, Rhodomonas sp. was cultivated in small 400 ml photobioreactors (Algaemist-S) under optimal conditions in turbidostat mode, which reached a biomass concentration of 1.5 gDW L–1 and dilution rate of 1.3 d–1. Samples were taken every 24 h for cell density and volume and productivity measurements in order to ensure a healthy and stable culture. In the next stage (N-starvation), the biomass was washed and transferred in a reactor filled with N-depleted medium. During N-starvation, samples were taken for biomass concentration, cell volume, PE and FA composition. The results of this study demonstrate that the lipid content increased significantly from 9% (t = 0 h) to 30% (t = 120 h) of the dry weight. After 120 h of N-starvation, the total FA content of Rhodomonas sp. remained stable for the remainder of the experiment (next 72 h). The highest increase of the FA concentration was represented by C16:0, C:18:1, C18:2, and C18:3, with highest concentrations after 120 h of starvation. The maximum EPA and DHA concentrations were observed after 48 h of starvation, while the maximum DHA to EPA ratio was detected at the end of the starvation.

Background Second messengers, c-di-GMP and (p)ppGpp, are vital regulatory molecules in bacteria, influencing cellular processes such as biofilm formation, transcription, virulence, quorum sensing, and proliferation. While c-di-GMP and (p)ppGpp are both synthesized from GTP molecules, they play antagonistic roles in regulating the cell cycle. In C. crescentus, c-di-GMP works as a major regulator of pole morphogenesis and cell development. It inhibits cell motility and promotes S-phase entry by inhibiting the activity of the master regulator, CtrA. Intracellular (p)ppGpp accumulates under starvation, which helps bacteria to survive under stressful conditions through regulating nucleotide levels and halting proliferation. (p)ppGpp responds to nitrogen levels through RelA-SpoT homolog enzymes, detecting glutamine concentration using a nitrogen phosphotransferase system (PTS Ntr). This work relates the guanine nucleotide-based second messenger regulatory network with the bacterial PTS Ntr system and investigates how bacteria respond to nutrient availability. Results We propose a mathematical model for the dynamics of c-di-GMP and (p)ppGpp in C. crescentus and analyze how the guanine nucleotide-based second messenger system responds to certain environmental changes communicated through the PTS Ntr system. Our mathematical model consists of seven ODEs describing the dynamics of nucleotides and PTS Ntr enzymes. Our simulations are consistent with experimental observations and suggest, among other predictions, that SpoT can effectively decrease c-di-GMP levels in response to nitrogen starvation just as well as it increases (p)ppGpp levels. Thus, the activity of SpoT (or its homologues in other bacterial species) can likely influence the cell cycle by influencing both c-di-GMP and (p)ppGpp. Conclusions In this work, we integrate current knowledge and experimental observations from the literature to formulate a novel mathematical model. We analyze the model and demonstrate how the PTS Ntr system influences (p)ppGpp, c-di-GMP, GMP and GTP concentrations. While this model does not consider all aspects of PTS Ntr signaling, such as cross-talk with the carbon PTS system, here we present our first effort to develop a model of nutrient signaling in C. crescentus.

Understanding the mechanisms of phenol production by microalgae can contribute to the development of microalgal biorefinery processes with higher economic and environmental sustainability. However, little is known about how phenols are produced and accumulate during microalgal cultivation. In this study, both extracellular and intracellular phenol production by two microalgal strains (Tetradesmus obliquus and Chlorella sp.) were investigated throughout a conventional photoautotrophic batch cultivation. The highest intracellular phenol content (10−25 mg g−1) and productivity (12–18 mg L−1 d−1) were attained for both strains in the first part of the batch, indicating a positive relation with nutrient availability and biomass productivity. Extracellular phenol production was 2–20 fold lower than intracellular phenols, but reached up to 27 mg L−1 for T. obliquus and 13 mg L−1 for Chlorella sp. The latter finding highlights relevant issues about the management of the exhausted culture medium, due to likely antimicrobial effects.

Thylakoid membranes, the site of photochemical and electron transport reactions of oxygenic photosynthesis, are composed of a myriad of proteins, cofactors including pigments, and glycerolipids. In the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803, the size and function of thylakoid membranes are reduced under nitrogen (N) starvation but are quickly recovered after N addition to the starved cells. To understand how the functionality of thylakoid membranes is adjusted in response to N status in Synechocystis sp. PCC 6803, we examined changes in thylakoid components and the photosynthetic activity during the N starvation and recovery processes. In N-starved cells, phycobilisome content, photosystem II protein levels and the photosynthetic activity substantially decreased as compared with those in N-sufficient cells. Although the content of chlorophyll (Chl) a, total protein and total glycerolipid also decreased under the N-starved condition based on OD730 reflecting cell density, when based on culture volume, the Chl a and total protein content remained almost constant and total glycerolipid content even increased during N starvation, suggesting that cellular levels of these components decrease under the N-starved condition mainly through dilution due to cell growth. With N addition, the photosynthetic activity quickly recovered, followed by full restoration of photosynthetic pigment and protein levels. The content of phosphatidylglycerol (PG), an essential lipid constituent of both photosystems, increased faster than that of Chl a, whereas the content of glycolipids, the main constituents of the thylakoid lipid bilayer, gradually recovered after N addition. The data indicate differential regulation of PG and glycolipids during the construction of the photosynthetic machinery and regeneration of thylakoid membranes. Of note, addition of PG to the growth medium slightly accelerated the Chl a accumulation in wild-type cells during the recovery process. Because PG is required for the biosynthesis of Chl a and the formation of functional photosystem complexes, rapid PG biosynthesis in response to N acquisition may be required for the rapid formation of the photosynthetic machinery during thylakoid regeneration.

Nitrogen (N) is probably the most important macronutrient and its scarcity limits plant growth, development and fitness. N starvation response has been largely studied by transcriptomic analyses, but little is known about the role of alternative polyadenylation (APA) in such response. In this work, we show that N starvation modifies poly(A) usage in a large number of transcripts, some of them mediated by FIP1, a component of the polyadenylation machinery. Interestingly, the number of mRNAs isoforms with poly(A) tags located in protein-coding regions or 5&prime, UTRs significantly increases in response to N starvation. The set of genes affected by APA in response to N deficiency is enriched in N-metabolism, oxidation-reduction processes, response to stresses, and hormone responses, among others. A hormone profile analysis shows that the levels of salicylic acid (SA), a phytohormone that reduces nitrate accumulation and root growth, increase significantly upon N starvation. Meta-analyses of APA-affected and fip1-2-deregulated genes indicate a connection between the nitrogen starvation response and salicylic acid (SA) signaling. Genetic analyses show that SA may be important for preventing the overgrowth of the root system in low N environments. This work provides new insights on how plants interconnect different pathways, such as defense-related hormonal signaling and the regulation of genomic information by APA, to fine-tune the response to low N availability.

In this research, the gene expression of genes involved in lipid metabolism of the eustigmatophyte alga Nannochloropsis gaditana was measured by transcriptomic data. This microalga can be used as a source of triacylglycerol (TAG) and the omega-3 fatty acid eicosapentaenoic acid (EPA). Insight in TAG and EPA production and regulation are needed to improve their productivity. Nitrogen starvation induces TAG accumulation in N. gaditana. Previous research showed that during nitrogen starvation, EPA was translocated from the polar lipids to TAG and de novo synthesized in N. gaditana. Therefore, the expression levels of genes involved in fatty acid translocation and de novo TAG synthesis were measured. Furthermore, the genes involved in de novo EPA synthesis such as elongases and desaturases were studied. The expression levels were measured during the first hours of nitrogen starvation and the subsequent period of 14 days. One phospholipid:diacylglycerol acyltransferase (PDAT) gene involved in translocation of fatty acids from membrane lipids to TAG was upregulated. In addition, several lipases were upregulated, suggesting that these enzymes might be responsible for the translocation of EPA to TAG. Most desaturases and elongases involved in de novo EPA synthesis were downregulated during nitrogen starvation, except for Δ9 desaturase which was upregulated. This upregulation correlates with the increase in oleic acid. Due to the presence of many hypothetical genes, improvement in annotation is needed to increase our understanding of these pathways and their regulation.

Cyanobacteria unable to fix atmospheric nitrogen have evolved sophisticated adaptations to survive to long periods of nitrogen starvation. These genetic programs are still largely unknown¿as evidenced by the many proteins whose expression is regulated in response to nitrogen availability, but which belong to unknown or hypothetical categories. In Synechocystis sp. PCC 6803, the global nitrogen regulator NtcA activates the expression of the sll0944 gene upon nitrogen deprivation. This gene encodes a protein that is highly conserved in cyanobacteria, but of unknown function. Based on the results described herein, we named the product of sll0944 carbon flow regulator A (CfrA). We analyzed the phenotypes of strains containing different levels of CfrA, including a knock-out strain (DcfrA), and two strains overexpressing CfrA from either the constitutive Ptrc promoter (Ptrc-cfrA) or the arsenite-inducible promoter ParsB (Pars-cfrA). Our results show that the amount of CfrA determines the accumulation of glycogen, and affects the synthesis of protein and photosynthetic pigments as well as amino acid pools. Strains with high levels of CfrA present high levels of glycogen and a decrease in photosynthetic pigments and protein content when nitrogen is available. Possible interactions between CfrA and the pyruvate dehydrogenase complex or PII protein have been revealed. The phenotype associated with CfrA overexpression is also observed in PII-deficient strains; however, it is lethal in this genetic background. Taken together, our results indicate a role for CfrA in the adaptation of carbon flux during acclimation to nitrogen deficiency.

The oleaginous microalga Ettlia oleoabundans accumulates both starch and lipids to high levels under stress conditions such as nitrogen starvation (N−). To steer biosynthesis towards starch or lipids only, it is important to understand the regulatory mechanisms involved. Here physiological and transcriptional changes under nitrogen starvation were analysed in controlled flat-panel photobioreactors at both short and long time-scales. Starch accumulation was transient and occurred rapidly within 24 h upon starvation, while lipid accumulation was gradual and reached a maximum after 4 days. The major fraction of accumulated lipids was composed of de novo synthesized neutral lipids - triacylglycerides (TAG) - and was characterized by a decreased composition of the polyunsaturated fatty acids (PUFAs) C18:3 and C16:3 and an increased composition of the mono-unsaturated (MUFAs) and saturated (SFAs) fatty acids C18:1/C16:1 and C18:0/C16:0, respectively. RNA-sequencing revealed that starch biosynthesis and degradation genes show different expression dynamics from lipid biosynthesis ones. An immediate rapid increase in starch synthetic transcripts was followed by an increase in starch degrading transcripts and a decrease in the starch synthetic ones. In contrast, increased gene expression for fatty acid and TAG synthesis was initiated later and occurred more gradually. Expression of several fatty acid desaturase (FAD) genes was decreased upon starvation, which corresponds to the observed changes to higher levels of MUFAs and SFAs. Moreover, several homologs of transcription regulators that were implicated in controlling starch and lipid metabolism in other microalgae showed differential gene expression and might be key regulators of starch and lipid metabolism in E. oleoabundans as well. Our data provide insights into the genetic foundation of starch and lipid metabolism in E. oleoabundans under nitrogen starvation and should facilitate metabolic engineering towards tailored strains with desired storage compound composition. Fil: Sturme, Mark H.J.. University of Agriculture Wageningen; Países Bajos Fil: Gong, Yanhai. Qingdao Institute Of Bioenergy And Bioprocess Technolog; China Fil: Heinrich, Josué Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Klok, Anne J.. University of Agriculture Wageningen; Países Bajos Fil: Eggink, Gerrit. University of Agriculture Wageningen; Países Bajos Fil: Wang, Dongmei. Qingdao Institute Of Bioenergy And Bioprocess Technolog; China Fil: Xu, Jian. Qingdao Institute Of Bioenergy And Bioprocess Technolog; China Fil: Wijffels, Rene H.. University of Agriculture Wageningen; Países Bajos

Bacterial response to environmental stimuli is essential for survival. In response to fluctuating environmental conditions, the physiological status of bacteria can change due to the actions of transcriptional regulatory machinery. The synthesis and accumulation of polyhydroxyalkanoates (PHAs) are one of the survival strategies in harsh environments. In this study, we used transcriptome analysis of Pseudomonas putida KT2440 to gain a genome-wide view of the mechanisms of environmental-friendly biopolymers accumulation under nitrogen-limiting conditions during conversion of metabolically different carbon sources (sodium gluconate and oleic acid). Transcriptomic data revealed that phaG expression is associated with medium-chain-length-PHAs’ synthesis not only on sodium gluconate but also on oleic acid, suggesting that PhaG may play a role in this process, as well. Moreover, genes involved in the β-oxidation pathway were induced in the PHAs production phase when sodium gluconate was supplied as the only carbon and energy source. The transition from exponential growth to stationary phase caused a significant expression of genes involved in nitrogen metabolism, energy supply, and transport system. In this study, several molecular mechanisms, which drive mcl-PHAs synthesis, have been investigated. The identified genes may provide valuable information to improve the efficiency of this bioprocess and make it more economically feasible. Electronic supplementary material The online version of this article (10.1007/s10295-018-2042-4) contains supplementary material, which is available to authorized users.

Much of our current knowledge of microbial growth is obtained from studies at a population level. Driven by the realization that processes that operate within a population might influence a population's behavior, we sought to better understand Tetradesmus obliquus (formerly Scenedesmus obliquus) physiology at the cellular level. In this work, an accurate pretreatment method to quantitatively obtain single cells of T. obliquus, a coenobia-forming alga, is described. These single cells were examined by flow cytometry for triacylglycerol (TAG), chlorophyll, and protein content, and their cell sizes were recorded by coulter counter. We quantified heterogeneity of size and TAG content at single-cell level for a population of T. obliquus during a controlled standard batch cultivation. Unexpectedly, variability of TAG content per cell within the population increased throughout the batch run, up to 400 times in the final stage of the batch run, with values ranging from 0.25 to 99 pg · cell-1 . Two subpopulations, classified as having low or high TAG content per cell, were identified. Cell size also increased during batch growth with average values from 36 to 70 μm3 · cell-1 ; yet cell size variability increased only up to 16 times. Cell size and cellular TAG content were not correlated at the single-cell level. Our data show clearly that TAG production is affected by cell-to-cell variation, which suggests that its control and better understanding of the underlying processes may improve the productivity of T. obliquus for industrial processes such as biodiesel production.

Microalgae are a sustainable source of lipids. A commonly used strategy for lipid accumulation in microalgae is a two-step batch cultivation, with a growth phase followed by a nitrogen starvation phase. A problem with this process is the decrease in photosynthetic efficiency during the nitrogen starvation phase, which leads to low lipid productivities. In this research, a new process strategy was studied with the aim to improve lipid productivity of the microalgae Nannochloropsis gaditana. The nitrogen concentrations were chosen to assure consumption of most part of the nitrogen during the night. An improvement of the photosystem II maximum quantum yield and an increase in the dry weight and TAG concentration was achieved from day 7 of nitrogen starvation onwards when the culture was fed with nitrogen each night compared to a culture without nitrogen addition. Consequently, the time-average TAG yield on light was also higher after 7 days of nitrogen starvation. However, since the maximal time-averaged triacylglycerol (TAG) yield on light was reached after 3 days of nitrogen starvation, the improved photosynthetic activity did not lead to an increase of the maximal time-averaged TAG yield on light. The culture with nitrogen addition had a higher protein concentration (1.1 compared to 0.7 g L−1), showing that the added nitrogen was mainly used for protein production. A higher chlorophyll a content (2.0 compared to 0.8 μg mg−1) showed improved photosystem and that a small part of nitrogen was used for chlorophyll a. Small nightly nitrogen additions during batch cultivation of nitrogen starved N. gaditana did result in improvement in photosystem II maximal quantum yield, biomass concentration, TAG production and a higher time-averaged maximal TAG yield on light, after 7 days of nitrogen starvation.

Triacylglycerol (TAG) accumulation in the microalgae Nannochloropsis gaditana is induced by nitrogen starvation and dependent on the light supplied. We studied under simulated outdoor light conditions the effect of supplied light on the TAG yield by varying the biomass-specific photon supply rate present at the onset of nitrogen starvation. High, intermediate and low average biomass-specific photon supply rates (26, 11 and 6 μmol g−1 s−1) were achieved by applying equal incident light intensity to different biomass concentrations (1.2, 2.9 and 5.4 g L−1). The intermediate biomass-specific photon supply rate resulted in the highest time-averaged TAG yield on light; 0.09 gTAG molph−1. Sub-optimal yields were attributed to photosaturation, photoinhibition, light falling through reactor without being absorbed and high maintenance requirements. The biomass-specific photon supply rate is important to optimize TAG production by microalgae. publishedVersion

The maintenance of adaptability to the exposure to agroecological extreme environments is generally a feature after the long-term domestication of crops. Auxin influences plant growth in all environments. At present, the research on the auxin response factors (ARFs) has been very in-depth. However, there is still a large gap in the research on the origin, evolution, and regulatory networks of the Auxin-responsive protein (AUX/IAA) family. Here, we identified 495 AUX/IAAs from 19 representative species covering aquatic algae to angiosperms and found that they originated from early bryophytes and mainly expanded by polyploidy in angiosperms. In the domesticated crop quinoa, the evolutionary model of the IAA family is relatively independent and forms a robust regulatory network with auxin signals and energy metabolism pathways. In the nitrogen-deficient environment, CqIAAs (Chenopodium quinoa AUX/IAAs), auxin signals, and TCA pathway genes induced expression in young roots to promote root elongation and could regulate the balance of carbon and nitrogen metabolism to maintain the adaptation of early seedlings in poor environments. Furthermore, a rapidly evolving CqIAA (AUR62011942) not only has different expression levels in two quinoa seeds but also has a significant stress response when seedlings face nitrogen deficiency stress, which may be a key factor in the adaptive regulation of the barren environment. Our research provides valuable clues for understanding the origin, evolution, and functional innovation of auxin signaling and also provides a reference for future agricultural breeding in the context of global environmental changes.

Autophagy is a conserved degradation pathway for recycling damaged organelles and aberrant proteins, and its important roles in plant adaptation to nutrient starvation have been generally reported. Previous studies found that overexpression of autophagy-related (ATG) gene MdATG10 enhanced the autophagic activity in apple roots and promoted their salt tolerance. The MdATG10 expression was induced by nitrogen depletion condition in both leaves and roots of apple plants. This study aimed to investigate the differences in the growth and physiological status between wild type and MdATG10-overexpressing apple plants in response to nitrogen starvation. A hydroponic system containing different nitrogen levels was used. The study found that the reduction in growth and nitrogen concentrations in different tissues caused by nitrogen starvation was relieved by MdATG10 overexpression. Further studies demonstrated the increased root growth and the higher nitrogen absorption and assimilation ability of transgenic plants. These characteristics contributed to the increased uptake of limited nitrogen nutrients by transgenic plants, which also reduced the starvation damage to the chloroplasts. Therefore, the MdATG10-overexpressing apple plants could maintain higher photosynthetic ability and possess better growth under nitrogen starvation stress.

Background Fungi are constantly exposed to nitrogen limiting environments, and thus the efficient regulation of nitrogen metabolism is essential for their survival, growth, development and pathogenicity. To understand how the rice blast pathogen Magnaporthe oryzae copes with limited nitrogen availability, a global proteome analysis under nitrogen supplemented and nitrogen starved conditions was completed. Methods M. oryzae strain 70–15 was cultivated in liquid minimal media and transferred to media with nitrate or without a nitrogen source. Proteins were isolated and subjected to unfractionated gel-free based liquid chromatography-tandem mass spectrometry (LC-MS/MS). The subcellular localization and function of the identified proteins were predicted using bioinformatics tools. Results A total of 5498 M. oryzae proteins were identified. Comparative analysis of protein expression showed 363 proteins and 266 proteins significantly induced or uniquely expressed under nitrogen starved or nitrogen supplemented conditions, respectively. A functional analysis of differentially expressed proteins revealed that during nitrogen starvation nitrogen catabolite repression, melanin biosynthesis, protein degradation and protein translation pathways underwent extensive alterations. In addition, nitrogen starvation induced accumulation of various extracellular proteins including small extracellular proteins consistent with observations of a link between nitrogen starvation and the development of pathogenicity in M. oryzae. Conclusion The results from this study provide a comprehensive understanding of fungal responses to nitrogen availability.

International audience; This study aims to investigate the triacylglycerol (TAG) productivity of Parachlorella kessleri grown under continuous illumination and to investigate its metabolism in simulated day/night cycles in order to estimate the feasibility of a large-scale production in outdoor solar photobioreactors. The strain was chosen for its ability to accumulate large amounts of triacylglycerol during nitrogen starvation. Several protocols of nitrogen starvation were tested in continuous illumination as well as in simulated day/night cycles. Sudden and progressive nitrogen starvation conditions have enhanced the TAG concentration and productivity of P. kessleri reaching up to 48 dry wt% and 4.4 × 10 −3 kg m −2 day −1 , respectively. Microalgal cell metabolism was significantly affected by the day/night illumination cycles. The energy-rich compounds (TAGs and carbohydrates) were accumulated by P. kessleri during the photope-riods and partly consumed during the dark to sustain the microalgae vitality. This TAG oxidation ultimately led to a 26% decrease in TAG productivity in cultures exposed to day/night cycles compared to ones exposed to continuous illumination of equal 24-h average photon flux density. The results can dictate the optimal time for harvesting cells for recovering the largest amount of TAGs.

Microalgae are important sources of triacylglycerols (TAGs) and high-value compounds such as carotenoids and long-chain polyunsaturated fatty acids (LC-PUFAs). TAGs are feedstocks for biofuels or edible oils; carotenoids are used as pigments in the food and feed industries; and LC-PUFAs are beneficial for human health, being also key to the correct development of fish in aquaculture. Current trends in microalgal biotechnology propose the combined production of biofuels with high-value compounds to turn large-scale production of microalgal biomass into an economically feasible venture. As TAGs, carotenoids and LC-PUFAs are lipophilic biomolecules, they not only share biosynthetic precursors and storage sinks, but also their regulation often depends on common environmental stimuli. In general, stressful conditions favor carotenoid and TAGs biosynthesis, whereas the highest accumulation of LC-PUFAs is usually obtained under conditions promoting growth. However, there are known exceptions to these general rules, as a few species are able to accumulate LC-PUFAs under low light, low temperature or long-term stress conditions. Thus, future research on how microalgae sense, transduce and respond to environmental stress will be crucial to understand how the biosynthesis and storage of these lipophilic molecules are regulated. The use of high-throughput methods (e.g. fluorescent activated cell sorting) will provide an excellent opportunity to isolate triple-producers, i.e. microalgae able to accumulate high levels of LC-PUFAs, carotenoids and TAGs simultaneously. Comparative transcriptomics between wild type and tripleproducers could then be used to identify key gene products involved in the regulation of these biomolecules even in microalgal species not amenable to reverse genetics. This combined approach could be a major step towards a better understanding of the microalgal metabolism under different stress conditions. Moreover, the generation of triple-producers would be essential to raise the biomass value in a biorefinery setting and contribute to meet the world's rising demand for food, feed and energy. Foundation for Science and Technology (Portugal) through research programme [CCMAR/Multi/04326/2013] doctoral research grants [SFRH/BD/105541/2014, SFRH/BD/115325/2016] Spanish Ministry of Economy and Competitiveness [AGL2016-74866-C3-02] CEIMAR Nord University and Nordland County Government project Bioteknologi-en framtidsrettet noering info:eu-repo/semantics/publishedVersion

Autophagy is an efficient degradation system for maintaining cellular homeostasis when plants are under environmental stress. ATG9 is the only integral membrane protein within the core ATG machinery that provides a membrane source for autophagosome formation. In this study, we isolated an ATG9 homologs gene in apple, MdATG9, from Malus domestica. The analysis of its sequence, subcellular localization, promoter cis-elements, and expression patterns revealed the potential function of MdATG9 in response to abiotic stressors. Overexpression of MdATG9 in apple callus conferred enhanced tolerance to nitrogen depletion stress. During the treatment, other important MdATGs were expressed at higher levels in transgenic callus than in the wild type. Furthermore, more free amino acids and increased sucrose levels were found in MdATG9-overexpression apple callus compared with the wild type in response to nitrogen starvation, and the expression levels of MdNRT1.1, MdNRT2.5, MdNIA1, and MdNIA2 were all increased higher in transgenic lines. These data suggest that, as an important autophagy gene, MdATG9 plays an important role in the maintenance of amino acids and sugars in response to nutrient starvation in apple.

Phycobilisomes (PBSs) are large (3&ndash, 5 megadalton) pigment-protein complexes in cyanobacteria that associate with thylakoid membranes and harvest light primarily for photosystem II. PBSs consist of highly ordered assemblies of pigmented phycobiliproteins (PBPs) and linker proteins that can account for up to half of the soluble protein in cells. Cyanobacteria adjust to changing environmental conditions by modulating PBS size and number. In response to nutrient depletion such as nitrogen (N) deprivation, PBSs are degraded in an extensive, tightly controlled, and reversible process. In Synechococcus elongatus UTEX 2973, a fast-growing cyanobacterium with a doubling time of two hours, the process of PBS degradation is very rapid, with 80% of PBSs per cell degraded in six hours under optimal light and CO2 conditions. Proteomic analysis during PBS degradation and re-synthesis revealed multiple proteoforms of PBPs with partially degraded phycocyanobilin (PCB) pigments. NblA, a small proteolysis adaptor essential for PBS degradation, was characterized and validated with targeted mass spectrometry. NblA levels rose from essentially 0 to 25,000 copies per cell within 30 min of N depletion, and correlated with the rate of decrease in phycocyanin (PC). Implications of this correlation on the overall mechanism of PBS degradation during N deprivation are discussed.

Background Among all organisms tested, Haematococcus pluvialis can accumulate the highest levels of natural astaxanthin. Nitrogen starvation and high irradiance promote the accumulation of starch, lipid, and astaxanthin in H. pluvialis, yet their cell division is significantly retarded. Accordingly, adaptive regulatory mechanisms are very important and necessary to optimize the cultivation conditions enabling an increase in biomass; as well as promoting astaxanthin accumulation by H. pluvialis. To clarify the intrinsic mechanism of high-level astaxanthin and biomass accumulation in the newly isolated strain, H. pluvialis JNU35, nitrogen-sufficiency and nitrogen-depletion conditions were employed. Time-resolved comparative transcriptome analysis was also conducted by crossing the two-step culture process. Results In the present study, we report the overall growth and physiological, biochemical, and transcriptomic characteristics of H. pluvialis JNU35 in response to nitrogen variation. From eight sampling time-points (2 days, 4 days, 8 days, 10 days, 12 days, 14 days, 16 days, and 20 days), 25,480 differentially expressed genes were found. These genes included the significantly responsive unigenes associated with photosynthesis, astaxanthin biosynthesis, and nitrogen metabolic pathways. The expressions of all key and rate-limiting genes involved in astaxanthin synthesis were significantly upregulated. The photosynthetic pathway was found to be attenuated, whereas the ferredoxin gene was upregulated, which might activate the cyclic electron-transport chain as compensation. Moreover, the expressions of genes related to nitrogen transport and assimilation were upregulated. The expressions of genes in the proteasome pathway were also upregulated. In contrast, the chloroplasts and nonessential proteins were gradually degraded, activating the specific ornithine–urea cycle pathway. These changes may promote the sustained accumulation of astaxanthin and biomass. Conclusions To the best of our knowledge, this paper is the first to investigate the long-term differences of gene expression from two-step culture process in the astaxanthin producer, H. pluvialis JNU35. According to our results, β-carotene ketolase (bkt1 and bkt2) serves as the key enzyme regulating astaxanthin accumulation in H. pluvialis JNU35. The cyclic electron-transport chain and novel nitrogen metabolic process were used adaptively as the regulatory mechanism compensating for different levels of stress. The in-depth study of these metabolic pathways and related key genes can reveal the underlying relationship between cell growth and astaxanthin accumulation in H. pluvialis JNU35.

Attenuated total reflection–Fourier transform infrared (ATR–FTIR) spectroscopy is a simple, cheap, and fast method to collect chemical compositional information from microalgae. However, (semi)quantitative evaluation of the collected data can be daunting. In this work, ATR–FTIR spectroscopy was used to monitor changes of protein, lipid, and carbohydrate content in seven green microalgae grown under nitrogen starvation. Three statistical methods—univariate linear regression analysis (ULRA), orthogonal partial least squares (OPLS), and multivariate curve resolution-alternating least squares (MCR–ALS)—were compared in their ability to model and predict the concentration of these compounds in the biomass. OPLS was found superior, since it i) included all three compounds simultaneously, ii) explained variations in the data very well, iii) had excellent prediction accuracy for proteins and lipids, and acceptable for carbohydrates, and iv) was able to discriminate samples based on cultivation stage and type of storage compounds accumulated in the cells. ULRA models worked well for the determination of proteins and lipids, but carbohydrates could only be estimated if already determined protein contents were used for scaling. Results obtained by MCR–ALS were similar to ULRA, however, this method is considerably easier to perform and interpret than the more abstract statistical/chemometric methods. FTIR-spectroscopy-based models allow high-throughput, cost-effective, and rapid estimation of biomass composition of green microalgae.

The microalga Nannochloropsis gaditana is a natural producer of triacylglycerol (TAG) and the omega-3 fatty acid eicosapentaenoic acid (EPA). TAG accumulation is induced by nitrogen starvation. The biomass-specific photon supply rate used had an effect on EPA and TAG accumulation during nitrogen starvation as well as on the localization of EPA accumulation. Clear differences in TAG yield on light were found for different biomass-specific photon supply rates and light regimes during nitrogen starvation. De novo EPA synthesis or the translocation of EPA between lipid fractions might be limiting for EPA accumulation in TAG. Further studies are needed to fully understand EPA accumulation in TAG during nitrogen starvation. To elucidate the function of EPA in TAG nitrogen recovery, experiments are suggested. The overexpression of genes involved in de novo EPA synthesis and translocation is proposed to elucidate the exact metabolic routes involved in these processes during nitrogen starvation. This work addresses future opportunities to increase EPA accumulation.

Non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is synthesized by cyanobacteria, diatoms, and dinoflagellates, and is known to be a causative agent of human neurodegenerative diseases. Different phytoplankton organisms’ ability to synthesize BMAA could indicate the importance of this molecule in the interactions between microalgae in nature. We were interested in the following: what kinds of mechanisms underline BMAA’s action on cyanobacterial cells in different nitrogen supply conditions. Herein, we present a proteomic analysis of filamentous cyanobacteria Nostoc sp. PCC 7120 cells that underwent BMAA treatment in diazotrophic conditions. In diazotrophic growth conditions, to survive, cyanobacteria can use only biological nitrogen fixation to obtain nitrogen for life. Note that nitrogen fixation is an energy-consuming process. In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by using LC-MS/MS spectrometry. Among them, 123 proteins belonging to different functional categories were selected—due to their notable expression differences—for further functional analysis and discussion. The presented proteomic data evidences that BMAA treatment leads to very strong (up to 80%) downregulation of α (NifD) and β (NifK) subunits of molybdenum-iron protein, which is known to be a part of nitrogenase. This enzyme is responsible for catalyzing nitrogen fixation. The genes nifD and nifK are under transcriptional control of a global nitrogen regulator NtcA. In this study, we have found that BMAA impacts in a total of 22 proteins that are under the control of NtcA. Moreover, BMAA downregulates 18 proteins that belong to photosystems I or II and light-harvesting complexes, BMAA treatment under diazotrophic conditions also downregulates five subunits of ATP synthase and enzyme NAD(P)H-quinone oxidoreductase. Therefore, we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as starvation-inducible DNA-binding protein, four SOS-response enzymes, and DNA repair enzymes, nine stress-response enzymes, and four proteases. The presented data provide new leads into the ecological impact of BMAA on microalgal communities that can be used in future investigations.

Summary Flavodoxin (Fld) plays a pivotal role in photosynthetic microorganisms as an alternative electron carrier flavoprotein under adverse environmental conditions. Cyanobacterial Fld has been demonstrated to be able to substitute ferredoxin of higher plants in most electron transfer processes under stressful conditions. We have explored the potential of Fld for use in improving plant stress response in creeping bentgrass (Agrostis stolonifera L.). Overexpression of Fld altered plant growth and development. Most significantly, transgenic plants exhibited drastically enhanced performance under oxidative, drought and heat stress as well as nitrogen (N) starvation, which was associated with higher water retention and cell membrane integrity than wild‐type controls, modified expression of heat‐shock protein genes, production of more reduced thioredoxin, elevated N accumulation and total chlorophyll content as well as up‐regulated expression of nitrite reductase and N transporter genes. Further analysis revealed that the expression of other stress‐related genes was also impacted in Fld‐expressing transgenics. Our data establish a key role of Fld in modulating plant growth and development and plant response to multiple sources of adverse environmental conditions in crop species. This demonstrates the feasibility of manipulating Fld in crop species for genetic engineering of plant stress tolerance.

Translationally controlled tumor protein (TCTP) is a multifunctional and highly conserved protein from yeast to humans. Recently, its role in non-selective autophagy has been reported with controversial results in mammalian and human cells. Herein we examine the effect of Mmi1, the yeast ortholog of TCTP, on non-selective autophagy in budding yeast Saccharomyces cerevisiae, a well-established model system to monitor autophagy. We induced autophagy by nitrogen starvation or rapamycin addition and measured autophagy by using the Pho8&Delta, 60 and GFP-Atg8 processing assays in WT, mmi1&Delta, and in autophagy-deficient strains atg8&Delta, or atg1&Delta, Our results demonstrate that Mmi1 does not affect basal or nitrogen starvation-induced autophagy. However, an increased rapamycin-induced autophagy is detected in mmi1&Delta, strain when the cells enter the post-diauxic growth phase, and this phenotype can be rescued by inserted wild-type MMI1 gene. Further, the mmi1&Delta, cells exhibit significantly lower amounts of reactive oxygen species (ROS) in the post-diauxic growth phase compared to WT cells. In summary, our study suggests that Mmi1 negatively affects rapamycin-induced autophagy in the post-diauxic growth phase and supports the role of Mmi1/TCTP as a negative autophagy regulator in eukaryotic cells.

Algal biofuel production requires an input of synthetic nitrogen fertilizer. Fertilizer synthesized via the Haber-Bosch process produces CO2 as a waste byproduct and represents a substantial financial and energy investment. Reliance on synthetic fertilizer attenuates the environmental significance and economic viability of algae production systems. To lower fertilizer input, the waste streams of algal production systems can be recycled to provide alternative sources of nitrogen such as amino acids to the algae. The halophytic green alga Dunaliella viridis can use ammonium (NH4+) derived from the abiotic degradation of amino acids, and previously, supplementation of NH4+ from glutamine degradation was shown to support acceptable levels of growth and increased neutral lipid production compared to nitrate. To understand the effect of glutamine-released NH4+ on algae growth and physiology, metabolite levels, growth parameters, and transcript profiles of D. viridis cultures were observed in a time course after transition from media containing nitrate as a sole N source to medium containing glutamine, glutamate, or a N-depleted medium. Growth parameters were similar between glutamine (NH4+) and nitrate supplemented cultures, however, metabolite data showed that the glutamine supplemented cultures (NH4+) more closely resembled cultures under nitrogen starvation (N-depleted and glutamate supplementation). Neutral lipid accumulation was the same in nitrate and glutamine-derived NH4+ cultures. However, glutamine-derived NH4+ caused a transcriptional response in the immediate hours after inoculation of the culture. The strong initial response of cultures to NH4+ changed over the course of days to closely resemble that of nitrogen starvation. These observations suggest that release of NH4+ from glutamine was sufficient to maintain growth, but not high enough to trigger a cell transition to a nitrogen replete state. Comparative transcript profiling of the nitrogen-starved and nitrate-supplied cultures show an overall downregulation of fatty acid synthesis and a shift to starch synthesis and accumulation. The results indicate that a continuous, amino acid derived slow release of NH4+ to algae cultures could reduce the amount of synthetic nitrogen needed for growth, but optimization is needed to balance nitrogen starvation and cell division.

Background Although microalgal biofuels have potential advantages over conventional fossil fuels, high production costs limit their application in the market. We developed bio-flocculation and incubation methods for the marine alga, Nannochloropsis oceanica CCMP1779, and the oleaginous fungus, Mortierella elongata AG77, resulting in increased oil productivity. Results By growing separately and then combining the cells, the M. elongata mycelium could efficiently capture N. oceanica due to an intricate cellular interaction between the two species leading to bio-flocculation. Use of a high-salt culture medium induced accumulation of triacylglycerol (TAG) and enhanced the contents of polyunsaturated fatty acids (PUFAs) including arachidonic acid and docosahexaenoic acid in M. elongata. To increase TAG productivity in the alga, we developed an effective, reduced nitrogen-supply regime based on ammonium in environmental photobioreactors. Under optimized conditions, N. oceanica produced high levels of TAG that could be indirectly monitored by following chlorophyll content. Combining N. oceanica and M. elongata to initiate bio-flocculation yielded high levels of TAG and total fatty acids, with ~ 15 and 22% of total dry weight (DW), respectively, as well as high levels of PUFAs. Genetic engineering of N. oceanica for higher TAG content in nutrient-replete medium was accomplished by overexpressing DGTT5, a gene encoding the type II acyl-CoA:diacylglycerol acyltransferase 5. Combined with bio-flocculation, this approach led to increased production of TAG under nutrient-replete conditions (~ 10% of DW) compared to the wild type (~ 6% of DW). Conclusions The combined use of M. elongata and N. oceanica with available genomes and genetic engineering tools for both species opens up new avenues to improve biofuel productivity and allows for the engineering of polyunsaturated fatty acids. Electronic supplementary material The online version of this article (10.1186/s13068-018-1172-2) contains supplementary material, which is available to authorized users.

Highlight The homeostasis of biologically active cytokinins was the predominant leaf-inherent factor for winter oilseed rape cultivar-differences in nitrogen starvation-induced leaf senescence and thus nitrogen efficiency., Nitrogen (N) efficiency of winter oilseed rape (Brassica napus L.) line-cultivars (cvs.), defined as high grain yield under N limitation, has been primarily attributed to maintained N uptake during reproductive growth (N uptake efficiency) in combination with delayed senescence of the older leaves accompanied with maintained photosynthetic capacity (functional stay-green). However, it is not clear whether genotypic variation in N starvation-induced leaf senescence is due to leaf-inherent factors and/or governed by root-mediated signals. Therefore, the N-efficient and stay-green cvs. NPZ-1 and Apex were reciprocally grafted with the N-inefficient and early-senescing cvs. NPZ-2 and Capitol, respectively and grown in hydroponics. The senescence status of older leaves after 12 days of N starvation assessed by SPAD, photosynthesis and the expression of the senescence-specific cysteine protease gene SAG12-1 revealed that the stay-green phenotype of the cvs. NPZ-1 and Apex under N starvation was primarily under the control of leaf-inherent factors. The same four cultivars were submitted to N starvation for up to 12 days in a time-course experiment. The specific leaf contents of biologically active and inactive cytokinins (CKs) and the expression of genes involved in CK homeostasis revealed that under N starvation leaves of early-senescing cultivars were characterized by inactivation of biologically active CKs, whereas in stay-green cultivars synthesis, activation, binding of and response to biologically active CKs were favoured. These results suggest that the homeostasis of biologically active CKs was the predominant leaf-inherent factor for cultivar differences in N starvation-induced leaf senescence and thus N efficiency.

The Arabidopsis homologs of mammalian lipin, PAH1 and PAH2, are cytosolic phosphatidic acid phosphohydrolases that are involved in phospholipid biosynthesis and are essential for growth under phosphate starvation. Here, pah1 pah2 double- knockout mutants were found to be hypersensitive to nitrogen (N) starvation, whereas transgenic plants overexpressing PAH1 or PAH2 in the pah1 pah2 mutant background showed a similar growth phenotype as compared with wild type (WT) under N starvation. The chlorophyll content of pah1 pah2 was significantly lower than that of WT, whereas the chlorophyll content and photosynthetic activity of the transgenic plants were significantly higher than those of WT under N-depleted conditions. Membrane glycerolipid composition of the pah1 pah2 mutants showed a significant decrease in the mole percent of chloroplast lipids to other phospholipids, whereas membrane lipid composition did not differ between transgenic plants and WT plants. Pulse-chase labeling experiments using plants grown under N-depleted conditions showed that, in pah1 pah2 plants, the labeling percent of chloroplast lipids such as phosphatidylglycerol and monogalactosyldiacylglycerol in the total glycerolipids was significantly lower than in WT. Moreover, N starvation-induced degradation of chloroplast structure was enhanced in pah1 pah2 mutants, and the membrane structure was recovered by complementation with PAH1. Thus, PAH is involved in maintaining chloroplast membrane structure and is required for growth under N-depleted conditions.

International audience; Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002(CrACX2), a gene encoding a member of the acyl-CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl-CoAs into trans-2-enoyl-CoA and produced H 2 O 2. This result demonstrated that CrACX2 is a genuine acyl-CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) β-oxidation spiral. A fluorescent protein-tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA β-oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of β-oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant-type FA β-oxidation involving H 2 O 2-producing acyl-CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii.