Volume 7, Issue 2, Pages 323-335 (February 2014) Chromosome Scale Genome Assembly and Transcriptome Profiling of Nannochloropsis gaditana in Nitrogen Depletion  Elisa Corteggiani Carpinelli, Andrea Telatin, Nicola Vitulo, Claudio Forcato, Michela D’Angelo, Riccardo Schiavon, Alessandro Vezzi, Giorgio Mario Giacometti, Tomas Morosinotto, Giorgio Valle  Molecular Plant  Volume 7, Issue 2, Pages 323-335 (February 2014) DOI: 10.1093/mp/sst120 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Representation of N. gaditana Scaffolds. Scaffolds accounting for complete chromosomes are represented in green, orange bars represent scaffolds that contain only one telomere, and, finally, in gray, scaffolds that do not contain the telomeric signal. Bar sizes are proportional to the length of the scaffolds; scale bars are indicated in the figure. Molecular Plant 2014 7, 323-335DOI: (10.1093/mp/sst120) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Comparative Analysis of Nannochloropsis gaditana and Nannochloropsis oceanica Predicted Proteins Based on Protein Cluster Analysis. Venn diagram of the shared/unique protein clusters of four Nannochloropsis strains compared to each other. Molecular Plant 2014 7, 323-335DOI: (10.1093/mp/sst120) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Confocal Microscopy Images of –N and +N Cultures at Day 6 after Re-Suspension in Fresh Media. Chlorophyll autofluorescence is in red, while emission relative to neutral lipids and carotenoids is reported in yellow. In nitrogen-depleted cells, chloroplasts have significantly reduced volumes comparing to +N cells. Upon Nile red staining, it is evident that, in –N cells, lipid droplets account for a prominent fraction of the cell volume, as highlighted by the magnified image. It is interesting to notice that +N cells without staining present a small yellow spot (clearly visible in the magnified image), whose emission spectrum is consistent with that of carotenoids. The signal could account for the orange-red eyespot visible in many Eustigmatophytes. Molecular Plant 2014 7, 323-335DOI: (10.1093/mp/sst120) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Molecular Functions Primarily Affected by Nitrogen Deficiency. We performed a GO enrichment analysis using the Fisher exact test (FDR = 0.05) to assess the presence of molecular functions overrepresented in the list of up-regulated and down-regulated genes after 3 d of nitrogen deprivation. Length of the bars represents the extent of overrepresentation (% of genes attributed to each GO in the test set divided by the % of genes attributed to the same GO in the genomes). In the list of up-regulated genes, we find GOs related to nitrogen metabolism and to functions involving ATP degradation (e.g. import of metabolites). Functions related to photosynthesis, respiration, and organellar gene expression are the vast majority of GOs overrepresented in the list of down-regulated genes. Molecular Plant 2014 7, 323-335DOI: (10.1093/mp/sst120) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Schematic Representation of Cellular Metabolism Reorganization upon Nitrogen Deprivation, according to RNA-Seq Data. Genes related to controlled protein degradation are induced in nitrogen stress. A number of processes involved in nitrogen recycling are up-regulated and we suggest a hypothesis based on our data that a glutamine–glutamate shuffle may act as a central intermediary of amine groups’ exchange between degradative and biosynthetic pathways. Biosynthetic processes indeed are not tuned down. In response to nitrogen deprivation, cells reorganize their metabolism by degrading selectively unnecessary molecules and synthesizing new ones to allow survival even in the deficiency of a fundamental nutrient. Molecular Plant 2014 7, 323-335DOI: (10.1093/mp/sst120) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Model of Nannochloropsis Energy–Carbon Metabolism in Response to Nitrogen Deficiency based on Gene Expression Analysis. In response to nitrogen deprivation, energetic metabolism in the organelles is tuned down. When mitochondrial efficiency decreases, complete oxidation of glucose into CO2 also becomes inefficient and leads to accumulation of Acetyl-CoA and reduced NADH. Re-oxidation of the NAD pool is necessary for glycolysis to proceed. In nitrogen-sufficient conditions, reduced NADH is mainly oxidized through respiration to yield ATP, while a minor fraction of Acetyl-CoA and reduced NAD(P)H is used for fatty acids synthesis. When mitochondria are tuned down in response to stress, a conspicuous fraction of Acetyl-CoA and reduced NAD(P)H is probably used in fatty acids biosynthesis. Genes responsible for fatty acid synthesis are constantly expressed (at least at the mRNA level) and this latter equilibrium leads to lipid droplet accumulation inside the cell. P, photosynthesis; G, glycolysis; R, respiration; LS, lipid synthesis. Molecular Plant 2014 7, 323-335DOI: (10.1093/mp/sst120) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions