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Summary Autophagy is a conserved degradation pathway that regulates the clearance of paternal substrate at the early embryogenesis stage of animals. However, its mode of action is likely different in plants, which can regenerate through apomixis without fertilisation. Somatic embryogenesis (SE) is a unique plant process widely used for plant propagation and germplasm utilisation. Here, we studied citrus as an example and found a higher autophagic activity after SE initiation. Interestingly, amyloplasts were frequently found inside autophagosomes, whereas the inhibition of autophagy blocks amyloplasts/starch degradation and hinders somatic embryo formation. Furthermore, the consumption of storage lipids was faster in autophagy mutants, suggesting lipid metabolism is activated when starch utilisation is blocked. Exogenous application of autophagy‐inducing chemicals (e.g. spermidine) significantly promoted the formation of autophagosomes and increased SE efficiency, indicating a positive correlation between autophagy, energy metabolism, and somatic embryo formation in citrus. Taken together, our study unveils a pathway for the degradation of plant‐specific organelles and provides an effective approach for plant propagation. [ABSTRACT FROM AUTHOR]

In the yeast Saccharomyces cerevisiae, the absence of the pseudouridine synthase Pus3/Deg1, which modifies tRNA positions 38 and 39, results in increased lipid droplet (LD) content and translational defects. In addition, starvation‐like transcriptome alterations and induced protein aggregation were observed. In this study, we show that the deg1 mutant increases specific misreading errors. This could lead to altered expression of the main regulators of neutral lipid synthesis which are the acetyl‐CoA carboxylase (Acc1), an enzyme that catalyzes a key step in fatty acid synthesis, and its regulator, the Snf1/AMPK kinase. We demonstrate that upregulation of the neutral lipid content of LD in the deg1 mutant is achieved by a mechanism operating in parallel to the known Snf1/AMPK kinase‐dependent phosphoregulation of Acc1. While in wild‐type cells removal of the regulatory phosphorylation site (Ser‐1157) in Acc1 results in strong upregulation of triacylglycerol (TG), but not steryl esters (SE), the deg1 mutation more specifically upregulates SE levels. In order to elucidate if other lipid species are affected, we compared the lipidomes of wild type and deg1 mutants, revealing multiple altered lipid species. In particular, in the exponential phase of growth, the deg1 mutant shows a reduction in the pool of phospholipids, indicating a compromised capacity to mobilize acyl‐CoA from storage lipids. We conclude that Deg1 plays a key role in the coordination of lipid storage and mobilization, which in turn influences lipid homeostasis. The lipidomic effects in the deg1 mutant may be indirect outcomes of the activation of various stress responses resulting from protein aggregation. [ABSTRACT FROM AUTHOR]

Very-long-chain fatty acids (VLCFA) are involved in a number of important plant physiological functions. Disorders in the expression of genes involved in the synthesis of VLCFA lead to a number of phenotypic consequences, ranging from growth retardation to the death of embryos. The elongation of VLCFA in the endoplasmic reticulum (ER) is carried out by multiple elongase complexes with different substrate specificities and adapted to the synthesis of a number of products required for a number of metabolic pathways. The information about the enzymes involved in the synthesis of VLCFA with more than 26 atoms of Carbon is rather poor. Recently, genes encoding enzymes involved in the synthesis of both regular-length fatty acids and VLCFA have been discovered and investigated. Polyunsaturated VLCFA in plants are formed mainly by 20:1 elongation into new monounsaturated acids, which are then imported into chloroplasts, where they are further desaturated. The formation of saturated VLCFA and their further transformation into a number of aliphatic compounds included in cuticular waxes and suberin require the coordinated activity of a large number of different enzymes. [ABSTRACT FROM AUTHOR]

Very-long-chain fatty acids (VLCFA) are involved in a number of important plant physiological functions. Disorders in the expression of genes involved in the synthesis of VLCFA lead to a number of phenotypic consequences, ranging from growth retardation to the death of embryos. The elongation of VLCFA in the endoplasmic reticulum (ER) is carried out by multiple elongase complexes with different substrate specificities and adapted to the synthesis of a number of products required for a number of metabolic pathways. The information about the enzymes involved in the synthesis of VLCFA with more than 26 atoms of Carbon is rather poor. Recently, genes encoding enzymes involved in the synthesis of both regular-length fatty acids and VLCFA have been discovered and investigated. Polyunsaturated VLCFA in plants are formed mainly by 20:1 elongation into new monounsaturated acids, which are then imported into chloroplasts, where they are further desaturated. The formation of saturated VLCFA and their further transformation into a number of aliphatic compounds included in cuticular waxes and suberin require the coordinated activity of a large number of different enzymes.

Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal for the production. An example of synthesis strongly dependent on external conditions is the production of storage lipids, which typically requires a high carbon/nitrogen ratio. This requirement also limits the use of abundant nitrogen-rich materials, such as industrial protein by-products, as substrates for lipid production. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered A. baylyi ADP1 by deleting the gene aceA encoding for isocitrate lyase and overexpressing fatty acyl-CoA reductase Acr1 in the wax ester production pathway to allow redirection of carbon towards WEs. This strategy led to 3-fold improvement in yield (0.075 ​g/g glucose) and 3.15-fold improvement in titer (1.82 ​g/L) and productivity (0.038 ​g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract, and baker’s yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids.

Azaspiracids (AZAs) are marine biotoxins including a variety of analogues. Recently, novel AZAs produced by the Mediterranean dinoflagellate Azadinium dexteroporum were discovered (AZA-54, AZA-55, 3-epi-AZA-7, AZA-56, AZA-57 and AZA-58) and their biological effects have not been investigated yet. This study aimed to identify the biological responses (biomarkers) induced in mussels Mytilus galloprovincialis after the bioaccumulation of AZAs from A. dexteroporum. Organisms were fed with A. dexteroporum for 21 days and subsequently subjected to a recovery period (normal diet) of 21 days. Exposed organisms accumulated AZA-54, 3-epi-AZA-7 and AZA-55, predominantly in the digestive gland. Mussels’ haemocytes showed inhibition of phagocytosis activity, modulation of the composition of haemocytic subpopulation and damage to lysosomal membranes; the digestive tissue displayed thinned tubule walls, consumption of storage lipids and accumulation of lipofuscin. Slight genotoxic damage was also observed. No clear occurrence of oxidative stress and alteration of nervous activity was detected in AZA-accumulating mussels. Most of the altered parameters returned to control levels after the recovery phase. The toxic effects detected in M. galloprovincialis demonstrate a clear biological impact of the AZAs produced by A. dexteroporum, and could be used as early indicators of contamination associated with the ingestion of seafood.

Lipids comprise a large group of chemically heterogeneous compounds. The majority have fatty acids (FA) as part of their structure, making these compounds suitable tools to examine processes raging from cellular to macroscopic levels of organization. Among the multiple roles of FA, they have structural functions as constituents of phospholipids which are the “building blocks” of cell membranes; as part of neutral lipids FA serve as storage materials in cells; and FA derivatives are involved in cell signalling. Studies on FA and their metabolism are important in numerous research fields, including biology, bacteriology, ecology, human nutrition and health. Specific FA and their ratios in cellular membranes may be used as biomarkers to enable the identification of organisms, to study adaptation of bacterial cells to toxic compounds and environmental conditions and to disclose food web connections. In this review, we discuss the various roles of FA in prokaryotes and eukaryotes and highlight the application of FA analysis to elucidate ecological mechanisms. We briefly describe FA synthesis; analyse the role of FA as modulators of cell membrane properties and FA ability to store and supply energy to cells; and inspect the role of polyunsaturated FA (PUFA) and the suitability of using FA as biomarkers of organisms.

The ribosomal RNA (rRNA) biosynthesis is the most energy consuming process in all living cells and the majority of total transcription activity is dedicated for synthesizing rRNA. The cells may adjust the synthesis of rRNA with the availability of resources. rRNA is mainly synthesized by RNA polymerase I that is composed of 14 subunits. Deletion of RPA12, 14, 39 and 49 are viable. RPA12 is a very small protein (13.6 kDa), and the amount of protein in the cells is very high (12,000 molecules per cell), but the role of this protein is unknown in other cellular metabolic processes (Kulak et al., 2014 [1]). RPA12 consists of two zinc-binding domains and it is required for the termination of rRNA synthesis (Mullem et al., 2002 [2]). Deletions of RPA12 in Saccharomyces cerevisiae and Schizosaccharomyces pombe cause a conditional growth defect (Nogi et al., 1993 [3]). In S. pombe, C-terminal deletion behaves like wild-type (Imazawa et al., 2001 [4]). This prompted us to investigate in detail the physiological role of RPA12 in S. cerevisiae, we performed the microarray of rpa12∆ strain and deposited into Gene Expression Omnibus under GSE68731. The analysis of microarray data revealed that the expression of major cellular metabolism genes is high. The amino acid biosynthesis, nonpolar lipid biosynthesis and glucose metabolic genes are highly expressed. The analyses also revealed that the rpa12∆ cells have an uncontrolled synthesis of cell metabolites, so RPA12 could be a master regulator for whole cellular metabolism.

Freezing injury is a major factor limiting the geographical distribution of plant species and the growth and yield of crop plants. Plants from temperate climates are able to increase their freezing tolerance during exposure to low but non-freezing temperatures in a process termed cold acclimation. Damage to cellular membranes is the major cause of freezing injury in plants, and membrane lipid composition is strongly modified during cold acclimation. Forward and reverse genetic approaches have been used to probe the role of specific lipid-modifying enzymes in the freezing tolerance of plants. In the present paper we describe an alternative ecological genomics approach that relies on the natural genetic variation within a species. Arabidopsis thaliana has a wide geographical range throughout the Northern Hemisphere with significant natural variation in freezing tolerance that was used for a comparative analysis of the lipidomes of 15 Arabidopsis accessions using ultra-performance liquid chromatography coupled to Fourier-transform mass spectrometry, allowing the detection of 180 lipid species. After 14 days of cold acclimation at 4°C the plants from most accessions had accumulated massive amounts of storage lipids, with most of the changes in long-chain unsaturated triacylglycerides, while the total amount of membrane lipids was only slightly changed. Nevertheless, major changes in the relative amounts of different membrane lipids were also evident. The relative abundance of several lipid species was highly correlated with the freezing tolerance of the accessions, allowing the identification of possible marker lipids for plant freezing tolerance. [ABSTRACT FROM AUTHOR]

Seasonal variation in lipid reserves of Calanus finmarchicus between seven different sampling zones namely; Central, North and Southern Irminger Sea, East Greenland Sea with characteristic current of Atlantic and Polar origin, Iceland Shelf, and Raykjanes Ridge (RR) were determined using High Performance Liquid Chromatography equipped with an Evaporative Light Scattering Detector (HPLC-ELSD). Wax ester (WE) and Triglycerides (TG) were the main lipids classes in C. finmarchicus. Along sampling zones in both spring and summer, the highest concentration of total storage lipids (WE+TG) were in samples from Central Irminger Sea (CIS) and the lowest in samples from Southern Irminger Sea (SIS). WE, TG as well as, the total storage lipids were significantly different between sampling zones and seasons, with WE in spring being lower than those in summer, and TG being higher in spring than in summer. Thus storage lipids varied along sampling zones between seasons in the Irminger Sea. [ABSTRACT FROM AUTHOR]

A comparative study was carried out on the dynamics of lipid accumulation in developing seeds of three lupine species. Lupine seeds differ in lipid content; yellow lupine (Lupinus luteus L.) seeds contain about 6%, white lupine (Lupinus albus L.) 7–14%, and Andean lupine (Lupinus mutabilis Sweet) about 20% of lipids by dry mass. Cotyledons from developing seeds were isolated and cultured in vitro for 96 h on Heller medium with 60 mM sucrose (+S) or without sucrose (–S). Each medium was additionally enriched with 35 mM asparagine or 35 mM NaNO3. Asparagine caused an increase in protein accumulation and simultaneously decreased the lipid content, but nitrate increased accumulation of both protein and lipid. Experiments with [1-14C]acetate and [2-14C]acetate showed that the decrease in lipid accumulation in developing lupine seeds resulted from exhaustion of lipid precursors rather than from degradation or modification of the enzymatic apparatus. The carbon atom from the C-1 position of acetate was liberated mainly as CO2, whereas the carbon atom from the C-2 position was preferentially used in anabolic pathways. The dominant phospholipid in the investigated lupine seed storage organs was phosphatidylcholine. The main fatty acid in yellow lupine cotyledons was linoleic acid, in white lupine it was oleic acid, and in Andean lupine it was both linoleic and oleic acids. The relationship between stimulation of lipid and protein accumulation by nitrate in developing lupine cotyledons and enhanced carbon flux through glycolysis caused by the inorganic nitrogen form is discussed. [ABSTRACT FROM PUBLISHER]

Deterioration of conifer seeds during prolonged storage has a negative impact on reforestation and gene conservation efforts. Western redcedar (Thuja plicata Donn ex D. Don) is a species of tremendous value to the forest industry. The seeds of this species are particularly prone to viability losses during long-term storage. Reliable tools to assess losses in seed viability during storage and their underlying causes, as well as the development of methods to prevent storage-related deterioration of seeds are needed by the forest industry. In this work, various imaging methods and biochemical analyses were applied to study deterioration of western redcedar seeds. Seedlots that exhibited poor germination performance, i.e. those that had experienced the greatest losses of viability during prolonged storage, exhibited greater abundance of oxidized proteins, detected by protein oxidation assays, and more pronounced changes in their in vivo 13C NMR spectra, most likely due to storage oil oxidation. The proportion of oxidized proteins also increased when seeds were subjected to accelerated ageing treatments. Detection of oxidized oils and proteins may constitute a reliable and useful tool for the forest industry. [ABSTRACT FROM PUBLISHER]

High-resolution 13C MAS NMR spectroscopy was used to profile a range of primary and secondary metabolites in vivo in intact whole seeds of eight different conifer species native to North America, including six of the Pinaceae family and two of the Cupressaceae family. In vivo 13C NMR provided information on the total seed oil content and fatty acid composition of the major storage lipids in a non-destructive manner. In addition, a number of monoterpenes were identified in the 13C NMR spectra of conifer seeds containing oleoresin; these compounds showed marked variability in individual seeds of Pacific silver fir within the same seed lot. In imbibed conifer seeds, the 13C NMR spectra showed the presence of considerable amounts of dissolved sucrose presumed to play a protective role in the desiccation-tolerance of seeds. The free amino acids arginine and asparagine, generated as a result of storage protein mobilization, were detected in vivo during seed germination and early seedling growth. The potential for NMR to profile metabolites in a non-destructive manner in single conifer seeds and seed populations is discussed. It is a powerful tool to evaluate seed quality because of its ability to assess reserve accumulation during seed development or at seed maturity; it can also be used to monitor reserve mobilization, which is critical for seedling emergence. [ABSTRACT FROM PUBLISHER]

Hatchling Cyprinodon variegatus were raised in the presence or absence of sediments contaminated with mixed trace elements to examine lethal and sublethal bioenergetic effects (metabolic rate, lipid storage, growth, reproduction) over a full life cycle (>1 year). Contaminated sediments were derived from a site receiving coal combustion residues (CCR) and were elevated in numerous trace elements including Al, As, Ba, Cd, Cu, Se, and V. Exposures were conducted at two levels of salinity (5 and 36 ppt) to examine the potential interaction of this variable with contaminants. Salinity had no effect on responses measured. Over the course of the study, fish exposed to contaminated sediment accumulated several CCR-related trace elements, including As, Cd, Se, and V. There were no differences in fish survival for contaminated sediment treatments and uncontaminated sediment treatments, nor were there differences in metabolic expenditures. However, growth, male condition factor, and storage lipid content in females were reduced due to contaminant exposure. No significant effects on fecundity or the proportion of females that were gravid at the end of the study were observed, yet females raised under control conditions produced 12% larger eggs than did females raised on contaminated sediments. During the presumably most-sensitive early life stages, individuals were not noticeably affected by contaminants, but rather the effects of exposure became apparent later in life. Because many species inhabit contaminated areas for long periods of time, often encompassing the entire life cycle, exposures focused only on specific life stages may substantially underestimate the overall responses elicited by individuals. [Copyright &y& Elsevier]

Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal for the production. An example of synthesis strongly dependent on external conditions is the production of storage lipids, which typically requires a high carbon/nitrogen ratio. This requirement also limits the use of abundant nitrogen-rich materials, such as industrial protein by-products, as substrates for lipid production. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered A. baylyi ADP1 by deleting the gene aceA encoding for isocitrate lyase and overexpressing fatty acyl-CoA reductase Acr1 in the wax ester production pathway to allow redirection of carbon towards WEs. This strategy led to 3-fold improvement in yield (0.075 ​g/g glucose) and 3.15-fold improvement in titer (1.82 ​g/L) and productivity (0.038 ​g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract, and baker's yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Azaspiracids (AZAs) are marine biotoxins including a variety of analogues. Recently, novel AZAs produced by the Mediterranean dinoflagellate Azadinium dexteroporum were discovered (AZA-54, AZA-55, 3-epi-AZA-7, AZA-56, AZA-57 and AZA-58) and their biological effects have not been investigated yet. This study aimed to identify the biological responses (biomarkers) induced in mussels Mytilus galloprovincialis after the bioaccumulation of AZAs from A. dexteroporum. Organisms were fed with A. dexteroporum for 21 days and subsequently subjected to a recovery period (normal diet) of 21 days. Exposed organisms accumulated AZA-54, 3-epi-AZA-7 and AZA-55, predominantly in the digestive gland. Mussels' haemocytes showed inhibition of phagocytosis activity, modulation of the composition of haemocytic subpopulation and damage to lysosomal membranes; the digestive tissue displayed thinned tubule walls, consumption of storage lipids and accumulation of lipofuscin. Slight genotoxic damage was also observed. No clear occurrence of oxidative stress and alteration of nervous activity was detected in AZA-accumulating mussels. Most of the altered parameters returned to control levels after the recovery phase. The toxic effects detected in M. galloprovincialis demonstrate a clear biological impact of the AZAs produced by A. dexteroporum, and could be used as early indicators of contamination associated with the ingestion of seafood. [ABSTRACT FROM AUTHOR]

Lipids comprise a large group of chemically heterogeneous compounds. The majority have fatty acids (FA) as part of their structure, making these compounds suitable tools to examine processes raging from cellular to macroscopic levels of organization. Among the multiple roles of FA, they have structural functions as constituents of phospholipids which are the "building blocks" of cell membranes; as part of neutral lipids FA serve as storage materials in cells; and FA derivatives are involved in cell signalling. Studies on FA and their metabolism are important in numerous research fields, including biology, bacteriology, ecology, human nutrition and health. Specific FA and their ratios in cellular membranes may be used as biomarkers to enable the identification of organisms, to study adaptation of bacterial cells to toxic compounds and environmental conditions and to disclose food web connections. In this review, we discuss the various roles of FA in prokaryotes and eukaryotes and highlight the application of FA analysis to elucidate ecological mechanisms. We briefly describe FA synthesis; analyse the role of FA as modulators of cell membrane properties and FA ability to store and supply energy to cells; and inspect the role of polyunsaturated FA (PUFA) and the suitability of using FA as biomarkers of organisms. [ABSTRACT FROM AUTHOR]

The ribosomal RNA (rRNA) biosynthesis is the most energy consuming process in all living cells and the majority of total transcription activity is dedicated for synthesizing rRNA. The cells may adjust the synthesis of rRNA with the availability of resources. rRNA is mainly synthesized by RNA polymerase I that is composed of 14 subunits. Deletion of RPA12, 14, 39 and 49 are viable. RPA12 is a very small protein (13.6 kDa), and the amount of protein in the cells is very high (12,000 molecules per cell), but the role of this protein is unknown in other cellular metabolic processes (Kulak et al., 2014 [1]). RPA12 consists of two zinc-binding domains and it is required for the termination of rRNA synthesis (Mullem et al., 2002 [2]). Deletions of RPA12 in Saccharomyces cerevisiae and Schizosaccharomyces pombe cause a conditional growth defect (Nogi et al., 1993 [3]). In S. pombe, C-terminal deletion behaves like wild-type (Imazawa et al., 2001 [4]). This prompted us to investigate in detail the physiological role of RPA12 in S. cerevisiae, we performed the microarray of rpa12 ∆ strain and deposited into Gene Expression Omnibus under GSE68731. The analysis of microarray data revealed that the expression of major cellular metabolism genes is high. The amino acid biosynthesis, nonpolar lipid biosynthesis and glucose metabolic genes are highly expressed. The analyses also revealed that the rpa12 ∆ cells have an uncontrolled synthesis of cell metabolites, so RPA12 could be a master regulator for whole cellular metabolism.