GSE129245
Nannochloropsis metabolism during transition from quiescence to autotrophy.
Purpose: The marine microalgae Nannochloropsis oceanica (CCMP1779) is a  prolific producer of oil and is considered a viable and sustainable  resource for biofuel feedstocks. Nitrogen (N) availability has a strong impact on the physiological status and metabolism of microalgal cells. However the exact nature of this response is poorly understood. To fill this gap we performed transcriptomic profiling combined with cellular and molecular analyses of N. oceanica CCMP1779 during the transition from quiescence to autotrophy. Methods: Nannochloropsis cells (50 ml per sample) were harvested for RNA isolation during nitrogen deprivation (N-) and after nitrogen resupply (NR) at the following time points: 6, 12, 24, 48 and 72 h. For each time point two biological replicates were harvested. RNA was extracted using the E.Z.N.A® Plant RNA kit (OMEGA) according to the manufacturer's instructions. RNA quality was verified using the Bioanalyzer (Agilent). Single-end, 50 bp nucleotide sequences were acquired for each sample using an Illumina HiSeq 2500 (MSU-Research Technology Support Facility). RNA-Seq reads were trimmed using Trimmomatic (v0.32) with removal of leading and trailing low quality bases (below quality score 10 with a 4-base wide sliding window) as well as TruSeq Single End adaptors. The resulting reads were aligned to the N. oceanica CCMP1779 genome assembly (GSE36959) using STAR (2.3.0e) (Dobin et al., 2013) by only allowing unique mapping, a maximum of 4 mismatches per pair and a maximum intron length of 10,000 bp. The HTSeq (0.6.1) was used to generate a count table for all genes using the exon as the feature type as well as the intersect-nonempty mode so that only reads that were completely aligned to two genes are considered ambiguous. HTSeq count tables were used for differential expression analysis using DEseq2. Differential gene expression was examined using DESeq2. A generalized linear model was fit, and we tested on condition (deprivation or recovery) and the time:condition interaction with log2-fold changes greater than 1. Significant differentially expressed genes with p-values less than 0.01 were identified after Benjamini-Hochberg (BH) multiple testing correction. Differentially expressed genes were clustered based on expression using K-means clustering (k = 4) with the Pearsons correlation distance metric within MeV (4.8.1). Results: We showed that N deprivation-induced quiescence is accompanied by a strong reorganization of the photosynthetic apparatus and changes in the lipid homeostasis leading to accumulation of triacylglycerol (TAG). Cell cycle activation and re-establishment of photosynthetic activity observed in response to resupply of the growth medium with N were accompanied by a rapid degradation of TAG stored in lipid droplets (LDs). Beside observing  LD translocation into vacuoles we also provide  evidence for direct interaction between the lipid droplet surface protein (NoLDSP) and autophagy-related 8 (NoATG8) protein and show a role of microlipophagy in LD turnover in N. oceanica CCMP1779. Conclusions: This knowledge  is crucial not only for understanding the fundamental mechanisms controlling the cellular energy homeostasis in microalgal cells but also for the development of efficient strategies to achieve higher algal biomass and better microalgal lipid productivity. Overall design: Nannochloropsis mRNA profiles during during nitrogen deprivation (N-) and after nitrogen resupply (NR) at the following time points: 6, 12, 24, 48 and 72 h were generated by deep sequencing, in duplicate, using Illumina HiSeq 2500.