| Abstract: | ![]()
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.In plants, carbon and nitrogen are directed to specific tissues or structures in accordance with developmental programs. In contrast, unicellular algae flexibly direct carbon and nitrogen to various macromolecules associated with specific intracellular compartments to optimize growth under varying environmental conditions. The signals responsible for this optimization strategy are poorly understood. They clearly are not driven by a developmental program but rather, responses to environmental cues. For example, under optimal growth conditions, ∼40% of the photosynthetically fixed carbon in typical eukaryotic microalga is directed toward the synthesis of amino acids that ultimately are incorporated into proteins (1–3). Over 50 y ago, however, it was recognized that, when nitrogen limits growth, intermediate metabolism is altered, and many microalgae can accumulate storage lipids, mainly in the form of triacylglycerols (TAGs) (4–6). This phenomenon is especially pronounced in diatoms.Diatoms, a highly successful class of eukaryotic algae that rose to ecological prominence during the past 30 My (7), often form massive blooms under turbulent conditions when nutrient supplies are highly variable (8). The ability of these organisms to optimize their growth under such conditions requires coordination of intermediate metabolism of carbon and nitrogen (9, 10). To optimize their growth, the first priority of the cells is to assimilate nitrogen into proteins, which also requires reducing equivalents and carbon skeletons that are primarily supplied by the tricarboxylic acid (TCA) cycle. However, when nitrogen availability decreases, the sink for TCA cycle metabolites declines, and acetyl-CoA, the source of carbon for the cycle, can be shunted toward fatty acid (FA) biosynthesis. Therefore, under nitrogen stress, cellular protein content decreases, whereas storage lipids increase (11, 12). This phenomenon has led to the hypothesis that overexpression of genes involved in lipid biosynthesis may increase the flux of carbon toward lipids (13, 14). Although this phenomenon is well-known, the signals that trigger the process remain unresolved. Genetic manipulations of lipid production in the model diatom, Phaeodactylum tricornutum, are ambiguous. Although there is one report showing that an overexpression of a type II diacylglycerol acyltransferase (DGAT; ProtID 49462) involved in TAG biosynthesis increases the accumulation of natural lipids in P. tricornutum (15), there are several reports indicating that manipulating FA biosynthesis does not significantly affect rates of lipid production (13, 14, 16).Using biochemical, physiological, bioinformatic, and reverse genetic approaches, we examine here how a diatom remodels intermediate metabolism to rapidly respond to nitrogen stress and its resupply. Our results reveal how carbon is redirected toward lipid biosynthesis under nitrogen stress in P. tricornutum. |
