Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress

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.lipid | metabolism | stress | NR | RNAi

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…Elevated expression of NR cohort genes after the addition of NO 3 2 is consistent with earlier studies in Arabidopsis (Scheible et al, 1997;Wang et al, 2000), where the addition of NO 3 2 was linked to increases both in uptake rates for NO 3 2 and in expression of NR and the genes involved in N-assimilation ( Figures 7A and 7C). Induced expression of the cohort in response to N-stress is also in agreement with previously described results in Arabidopsis thaliana and P. tricornutum (Alipanah et al, 2015;Glass et al, 2002;Levitan et al, 2015;Scheible et al, 2004;Wang et al, 2000Wang et al, , 2004. In NR-KO14 cells, the patterns of gene expression were atypical.…”

“…(I) J33873 is also annotated as a PITP. et al, 2015; Levitan et al, 2015). Our analysis shows that the addition of NO 3 2 to NR-KO cells has an acute, immediate impact on key metabolic pathways such as N-assimilation and C-fixation, structural pathways for light-harvesting complexes, ribosome production, chlorophyll biosynthesis, and carbon partitioning.…”

“…Shortly after NO 3 2 is introduced to NR-KO14 cells, expression levels of key functional and metabolic gene clusters indirectly related to N-assimilation are sharply downregulated. Despite ample extra-and intracellular NO 3 2 , transcript profiles for these clusters respond similarly to those observed in wild-type cells under N-stress (Alipanah et al 2015;Bender et al, 2014;Juergens et al, 2015;Levitan et al, 2015;Schmollinger at al., 2014). However, in contrast to wild-type cells which gradually acclimate to a dwindling supply of N-product, the response in NR-KO14 cells is quite rapid.…”

“…Phosphatidic acid phosphatase (PAP, J39949), the penultimate enzyme in the TAG pathway, showed a spike in transcription at 18 h ( Figure 10E). Expression levels of J43469, diacylglycerol acyltransferase 2 (DGAT2), which completes the final step in TAG biosynthesis, although previously reported as strongly upregulated in N-stress conditions (Mus et al, 2013;Levitan et al, 2015), were only slightly higher in NR-KO14 cells (18 h) relative to the wild type ( Figure 10F). At 18 h, expression of the gene (J48859), a homolog in P. tricornutum of the major lipid droplet protein (MLDP), was ;5 times more highly expressed in the NR-KO14 transcriptome compared with the wild type ( Figure 10G); MLDP has been reported in other microalgae to regulate lipid droplet size (Yoneda et al, 2016).…”

“…In NO 3 2 -limited conditions, diatoms and certain green algae redirect C from storage carbohydrates such as chrysolaminarin and starch to accumulation of neutral lipids, specifically triacylglycerol (TAG) (Rismani-Yazdi et al, 2012;Shifrin and Chisholm, 1981;Yu et al, 2009). Much recent work has focused both on characterizing the genes and metabolic pathways that induce lipid biosynthesis and on identifying routes and regulators that could be harnessed to substantially increase TAG production without negatively affecting growth (Alipanah et al, 2015;Ge et al, 2014;Hildebrand et al, 2012;Levitan et al, 2015;Radakovits et al, 2010;Trentacoste et al, 2013;Yang et al, 2013;Yoon et al, 2012). Deleterious impacts of NO 3 2 depletion include reduction of photosynthetic capacity, C-fixation, and N-assimilation and cessation of growth (Alipanah et al, 2015;Bender et al, 2014;Hockin et al, 2012;Syrett et al, 1986;Turpin, 1991).…”