Diatoms are unicellular algae that accumulate significant amounts of triacylglycerols as storage lipids when their growth is limited by nutrients. Using biochemical, physiological, bioinformatics, and reverse genetic approaches, we analyzed how the flux of carbon into lipids is influenced by nitrogen stress in a model diatom, Phaeodactylum tricornutum. Our results reveal that the accumulation of lipids is a consequence of remodeling of intermediate metabolism, especially reactions in the tricarboxylic acid and the urea cycles. Specifically, approximately one-half of the cellular proteins are cannibalized; whereas the nitrogen is scavenged by the urea and glutamine synthetase/glutamine 2-oxoglutarate aminotransferase pathways and redirected to the de novo synthesis of nitrogen assimilation machinery, simultaneously, the photobiological flux of carbon and reductants is used to synthesize lipids. To further examine how nitrogen stress triggers the remodeling process, we knocked down the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The strain exhibits 40-50% of the mRNA copy numbers, protein content, and enzymatic activity of the wild type, concomitant with a 43% increase in cellular lipid content. We suggest a negative feedback sensor that couples photosynthetic carbon fixation to lipid biosynthesis and is regulated by the nitrogen assimilation pathway. This metabolic feedback enables diatoms to rapidly respond to fluctuations in environmental nitrogen availability.lipid | metabolism | stress | NR | RNAi

No abstract

Deschampsia antarctica Desv. (Poaceae) is the only grass that grows in the maritime Antarctic. Constant low temperatures and episodes of high light are typical conditions during the growing season at this latitude. These factors enhance the formation of active oxygen species and may cause photoinhibition. Therefore, an efficient mechanism of energy dissipation and / or scavenging of reactive oxygen species (ROS) would contribute to survival in this harsh environment. In this paper, non-acclimated and cold-acclimated D. antarctica were subjected to high light and / or low temperature for 24 h. The contribution of non-photochemical dissipation of excitation light energy and the activities of detoxifying enzymes in the development of resistance to chilling induced photoinhibition were studied by monitoring PSII fluorescence, total soluble antioxidants, and pigments contents and measuring variations in activity of superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11), and glutathione reductase (GR; EC 1.6.4.2). The photochemical efficiency of PSII, measured as Fv / F m, and the yield of PSII electron transport (ΦPSII) both decreased under high light and low temperatures. In contrast, photochemical quenching (qP) in both non-acclimated and cold-acclimated plants remained relatively constant (approximately 0.8) in high-light-treated plants. Unexpectedly, qP was lower (0.55) in cold-acclimated plants exposed to 4°C and low light intensity. Activity of SOD in cold-acclimated plants treated with high light at low temperature showed a sharp peak 2–4 h after the beginning of the experiment. In cold-acclimated plants APX remained high with all treatments. Activity of GR decreased in cold-acclimated plants. Compared with other plants, D. antarctica exhibited high levels of SOD and APX activity. Pigment analyses show that the xanthophyll cycle is operative in this plant. We propose that photochemical quenching and particularly the high level of antioxidants help D. antarctica to resist photoinhibitory conditions. The relatively high antioxidant capacity of D. antarctica may be a determinant for its survival in the harsh Antarctic environment.

SUMMARYWhen diatoms are stressed for inorganic nitrogen they remodel their intermediate metabolism and redirect carbon towards lipid biosynthesis. However, this response comes at a significant cost reflected in decreased photosynthetic energy conversion efficiency and growth. Here we explore a molecular genetics approach to restrict the assimilation of inorganic nitrogen by knocking down nitrate reductase (NR). The transformant strain, NR21, exhibited about 50% lower expression and activity of the enzyme but simultaneously accumulated over 40% more fatty acids. However, in contrast to nitrogen-stressed wild-type (WT) cells, which grow at about 20% of the rate of nitrogen-replete cells, growth of NR21 was only reduced by about 30%. Biophysical analyses revealed that the photosynthetic energy conversion efficiency of photosystem II was unaffected in NR21; nevertheless, the plastoquinone pool was reduced by 50% at the optimal growth irradiance while in the WT it was over 90% oxidized. Further analyses reveal a 12-fold increase in the glutamate/glutamine ratio and an increase NADPH and malonyl-CoA pool size. Transcriptomic analyses indicate that the knock down resulted in changes in the expression of genes for lipid biosynthesis, as well as the expression of specific transcription factors. Based on these observations, we hypothesize that the allocation of carbon and reductants in diatoms is controlled by a feedback mechanism between intermediate metabolites, the redox state of the plastid and the expression and binding of transcription factors related to stress responses.

Under nutrient deplete conditions, diatoms accumulate between 15% to 25% of their dry weight as lipids, primarily as triacylglycerols (TAGs). As in most eukaryotes, these organisms produce TAGs via the acyl-CoA dependent Kennedy pathway. The last step in this pathway is catalyzed by diacylglycerol acyltransferase (DGAT) that acylates diacylglycerol (DAG) to produce TAG. To test our hypothesis that DGAT plays a major role in controlling the flux of carbon towards lipids, we overexpressed a specific type II DGAT gene, DGAT2D, in the model diatom Phaeodactylum tricornutum. The transformants had 50- to 100-fold higher DGAT2D mRNA levels and the abundance of the enzyme increased 30- to 50-fold. More important, these cells had a 2-fold higher total lipid content and incorporated carbon into lipids more efficiently than the wild type (WT) while growing only 15% slower at light saturation. Based on a flux analysis using C as a tracer, we found that the increase in lipids was achieved via increased fluxes through pyruvate and acetyl-CoA. Our results reveal overexpression of DAGT2D increases the flux of photosynthetically fixed carbon towards lipids, and leads to a higher lipid content than exponentially grown WT cells.