High Resolution Proteome of Lipid Droplets Isolated from the Pennate Diatom Phaeodactylum tricornutum (Bacillariophyceae) Strain pt4 provides mechanistic insights into complex intracellular coordination during nitrogen deprivation.

Lipid droplets (LDs) are an organelle conserved amongst all eukaryotes, consisting of a neutral lipid core surrounded by a polar lipid monolayer. Many species of microalgae accumulate LDs in response to stress conditions, such as nitrogen starvation. Here, we report the isolation and proteomic profiling of LD proteins from the model oleaginous pennate diatom Phaeodactylum tricornutum, strain Pt4 (UTEX 646). We also provide a quantitative description of LD morphological ontogeny, and fatty acid content. Novel cell disruption and LD isolation methods, combined with suspension-trapping and nanoflow liquid chromatography coupled to high resolution mass spectrometry, yielded an unprecedented number of LD proteins. Predictive annotation of the LD proteome suggests a broad assemblage of proteins with diverse functions, including lipid metabolism and vesicle trafficking, as well as ribosomal and proteasomal machinery. These proteins provide mechanistic insights into LD processes, and evidence for interactions between LDs and other organelles. We identify for the first time several key steps in diatom LD-associated triacylglycerol biosynthesis. Bioinformatic analyses of the LD proteome suggests multiple protein targeting mechanisms, including amphipathic helices, post-translational modifications, and translocation machinery. This work corroborates recent findings from other strains of P. tricornutum, other diatoms, and other eukaryotic organisms, suggesting that the fundamental proteins orchestrating LDs are conserved, and represent an ancient component of the eukaryotic endomembrane system. We postulate a comprehensive model of nitrogen starvation-induced diatom LDs on a molecular scale, and provide a wealth of candidates for metabolic engineering, with the potential to eventually customize LD contents.

Long-Chain Polyunsaturated Fatty Acids in the Green Microalga Lobosphaera incisa Contribute to Tolerance to Abiotic Stresses.

Lobosphaera incisa is a green microalga that accumulates high levels of the valuable omega-6 long-chain polyunsaturated fatty acids (LC-PUFA) arachidonic acid (ARA, 20:4n-6) in triacylglycerols (TAG) under nitrogen (N) starvation. LC-PUFA accumulation is a rare trait in photosynthetic microalgae with insufficiently understood physiological significance. In this study, RNAi was attempted, for the first time in L. incisa, to produce knockdown lines for the Δ5 desaturase gene. Two lines, termed modified lines, which were isolated during screening for transgenic events, demonstrated alterations in their LC-PUFA profile, ARA-biosynthesis gene expression and lipid class distribution. In line M5-78, which appeared to carry a mutation in the Δ6 elongase gene, LC-PUFA were substituted by 18:3n-6 in all glycerolipids. Line M2-35, for which the exact genetic background has not been established, displayed a dramatic reduction in 20:4n-6, concomitant with an augmented proportion of 18:1n-9, in particular in the extraplastidial membrane lipids and TAG. The physiological responses of the modified lines to stressful conditions were compared with the wild type and the Δ5 desaturase mutant. In the N-replete cells of modified lines, the frequency of lipid droplets was reduced, while a number of starch grains increased, suggesting altered partitioning of assimilated carbon into reserve products. Furthermore, both lines exhibited reduced ability to accumulate TAG under N deprivation and recover from N starvation. Both lines demonstrated lower photosynthetic pigment contents, impairments in photosynthesis under a range of stressful conditions, and less efficient functioning of photoprotection under optimal conditions. Possible implications of fatty acids modifications in the stress response of L. incisa are addressed.

Arachidonic acid is important for efficient use of light by the microalga Lobosphaera incisa under chilling stress.

The oleaginous microalga Lobosphaera incisa (Trebouxiophyceae, Chlorophyta) contains arachidonic acid (ARA, 20:4 n-6) in all membrane glycerolipids and in the storage lipid triacylglycerol. The optimal growth temperature of the wild-type (WT) strain is 25°C; chilling temperatures (≤15°C) slow its growth. This effect is more pronounced in the delta-5-desaturase ARA-deficient mutant P127, in which ARA is replaced with dihomo-γ-linolenic acid (DGLA, 20:3 n-6). In nutrient-replete cells grown at 25°C, the major chloroplast lipid monogalactosylglycerol (MGDG) was dominated by C18/C16 species in both strains. Yet ARA constituted over 10% of the total fatty acids in the WT MGDG as a component of C20/C18 and C20/C20 species, whereas DGLA was only a minor component of MGDG in P127. Both strains increased the percentage of 18:3 n-3 in membrane lipids under chilling temperatures. The temperature downshift led to a dramatic increase in triacylglycerol at the expense of chloroplast lipids. WT and P127 showed a similarly high photochemical quantum yield of photosystem II, whereas non-photochemical quenching (NPQ) and violaxanthin de-epoxidation were drastically higher in P127, especially at 15°C. Fluorescence anisotropy measurements indicated that ARA-containing MGDG might contribute to sustaining chloroplast membrane fluidity upon dropping to the chilling temperature. We hypothesize that conformational changes in chloroplast membranes and increased rigidity of the ARA-deficient MGDG of P127 at chilling temperatures are not compensated by trienoic fatty acids. This might 'lock' violaxanthin de-epoxidase in the activated state causing high constitutive NPQ and alleviate the risk of photodamage under chilling conditions in the mutant.

Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii.

The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga.

Growth, lipid production and metabolic adjustments in the euryhaline eustigmatophyte Nannochloropsis oceanica CCALA 804 in response to osmotic downshift.

We investigated the effects of osmotic downshift induced by the transfer of Nannochloropsis oceanica CCALA 804 from artificial seawater medium (27 g L(-1) NaCl) to the same medium without NaCl or freshwater modified BG-11 medium (mBG-11) as a function of photosynthetically active radiation (170, 350, or 700 µmol photon m(-2) s(-1)). Alterations in growth, total fatty acid (FA) content and FA composition of individual lipid classes, and in relative contents of metabolites relevant to osmotic adjustments were studied. Cells displayed remarkable tolerance to the osmotic downshift apart from some swelling, with no substantial lag or decline in cell division rate. Biomass accumulation and chlorophyll a content were enhanced upon downshifting, especially under the highest irradiance. The highest chlorophyll a and eicosapentaenoic acid (EPA) biomass and culture contents were determined in the cultures grown in mBG-11. Two days after transfer to 0 g L(-1) NaCl, the proportion in total acyl lipids of the major chloroplast galactolipid monogalactosyldiacylglycerol, a major depot of EPA, increased twofold, along with a modest change in the proportion of digalactosyldiacylglycerol (DGDG). EPA percentage decreased in DGDG and increased in the extraplastidial lipid phosphatidylethanolamine. Metabolite profiling by GC-MS analysis revealed a sharp decrease in metabolites potentially involved in osmoregulation, such as mannitol and proline, while proline-cycle intermediates and some free sugars increased. The stress-induced polyamine spermidine decreased ca. one order of magnitude, while its catabolic product-the non-protein amino acid γ-amino butyric acid-increased twofold, as did the stress-related sugars trehalose and talose. Biochemical mechanisms governing osmotic plasticity and implications for optimization of EPA production by N. oceanica CCALA 804 under variable cultivation conditions are discussed.

Comparative Analysis of Volatiles Emitted from Tomato and Pepper Plants in Response to Infection by Two Whitefly-Transmitted Persistent Viruses.

The whitefly Bemisia tabaci is one of the most important agricultural pests due to its extreme invasiveness, insecticide resistance, and ability to transmit hundreds of plant viruses. Among these, Begomoviruses and recombinant whitefly-borne Poleroviruses are transmitted persistently. Several studies have shown that upon infection, plant viruses manipulate plant-emitted volatile organic compounds (VOCs), which have important roles in communication with insects. In this study, we profiled and compared the VOCs emitted by tomato and pepper plant leaves after infection with the Tomato yellow leaf curl virus (TYLCV) (Bogomoviruses) and the newly discovered Pepper whitefly-borne vein yellows virus (PeWBVYV) (Poleroviruses), respectively. The results identified shared emitted VOCs but also uncovered unique VOC signatures for each virus and for whitefly infestation (i.e., without virus infection) independently. The results suggest that plants have general defense responses; however, they are also able to respond individually to infection with specific viruses or infestation with an insect pest. The results are important to enhance our understanding of virus- and insect vector-induced alteration in the emission of plant VOCs. These volatiles can eventually be used for the management of virus diseases/insect vectors by either monitoring or disrupting insect-plant interactions.

Diversity and Differentiation of Duckweed Species from Israel.

Duckweeds (Lemnaceae) are tiny plants that float on aquatic surfaces and are typically isolated from temperate and equatorial regions. Yet, duckweed diversity in Mediterranean and arid regions has been seldom explored. To address this gap in knowledge, we surveyed duckweed diversity in Israel, an ecological junction between Mediterranean and arid climates. We searched for duckweeds in the north and center of Israel on the surface of streams, ponds and waterholes. We collected and isolated 27 duckweeds and characterized their morphology, molecular barcodes (atpF-atpH and psbK-psbI) and biochemical features (protein content and fatty acids composition). Six species were identified-Lemna minor, L. gibba and Wolffia arrhiza dominated the duckweed populations, and together with past sightings, are suggested to be native to Israel. The fatty acid profiles and protein content further suggest that diverged functions have attributed to different haplotypes among the identified species. Spirodela polyrhiza, W. globosa and L. minuta were also identified but were rarer. S. polyrhiza was previously reported in our region, thus, its current low abundance should be revisited. However, L. minuta and W. globosa are native to America and Far East Asia, respectively, and are invasive in Europe. We hypothesize that they may be invasive species to our region as well, carried by migratory birds that disperse them through their migration routes. This study indicates that the duckweed population in Israel's aquatic environments consists of both native and transient species.

Lipidome Remodeling and Autophagic Respose in the Arachidonic-Acid-Rich Microalga Lobosphaera incisa Under Nitrogen and Phosphorous Deprivation.

The green microalga Lobosphaera incisa accumulates triacylglycerols (TAGs) with exceptionally high levels of long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid (ARA) under nitrogen (N) deprivation. Phosphorous (P) deprivation induces milder changes in fatty acid composition, cell ultrastructure, and growth performance. We hypothesized that the resource-demanding biosynthesis and sequestration of ARA-rich TAG in lipid droplets (LDs) are associated with the enhancement of catabolic processes, including membrane lipid turnover and autophagic activity. Although this work focuses mainly on N deprivation, a comparative analysis of N and P deprivation responses is included. The results of lipidomic profiling showed a differential impact of N and P deprivation on the reorganization of glycerolipids. The formation of TAG under N deprivation was associated with the enhanced breakdown of chloroplast glycerolipids and the formation of lyso-lipids. N-deprived cells displayed a profound reorganization of cell ultrastructure, including internalization of cellular material into autophagic vacuoles, concomitant with the formation of LDs, while P-deprived cells showed better cellular ultrastructural integrity. The expression of the hallmark autophagy protein ATG8 and the major lipid droplet protein (MLDP) genes were coordinately upregulated, but to different extents under either N or P deprivation. The expression of the Δ5-desaturase gene, involved in the final step of ARA biosynthesis, was coordinated with ATG8 and MLDP, exclusively under N deprivation. Concanamycin A, the inhibitor of vacuolar proteolysis and autophagic flux, suppressed growth and enhanced levels of ATG8 and TAG in N-replete cells. The proportions of ARA in TAG decreased with a concomitant increase in oleic acid under both N-replete and N-deprived conditions. The photosynthetic apparatus's recovery from N deprivation was impaired in the presence of the inhibitor, along with the delayed LD degradation. The GFP-ATG8 processing assay showed the release of free GFP in N-replete and N-deprived cells, supporting the existence of autophagic flux. This study provides the first insight into the homeostatic role of autophagy in L. incisa and points to a possible metabolic link between autophagy and ARA-rich TAG biosynthesis.