Long Serial Analysis of Gene Expression for Gene Discovery and Transcriptome Profiling in the Widespread Marine Coccolithophore Emiliania huxleyi

Abstract

The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO 2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism.Coccolithophores are an abundant and widespread phytoplankton functional group responsible for significant amounts of calcification in the ocean. This group is intensively studied for its roles in the marine carbon and sulfur cycles, the production of alkenones, and marine calcification. The coccolithophore Emiliania huxleyi is the most abundant species of this functional group in the modern ocean, and it blooms in both coastal and open ocean regions (24). E. huxleyi both fixes CO 2 through photosynthesis and generates CO 2 through the biomineralization of calcium carbonate (calcification). Photosynthesis and calcification are important components of the global carbon (C) cycle. Ultimately, both the presence of E. huxleyi blooms and the ratio of photosynthesis to calcification within the population mediate exchange between atmospheric and oceanic CO 2 . As such, coccolithophores are being intensively studied for their role in the C cycle and their potential influence on global climate.Nitrogen (N) and phosphorus (P) are two critical macronutrients for E. huxleyi growth, and their availability can impact when and where E. huxleyi blooms are able to occur (20). Further, N and P starvation can influence CO 2 exchange by changing rates of photosynthesis and calcification (24). For example, P starvation typically increases calcifi...

Metatranscriptome analyses indicate resource partitioning between diatoms in the field

“…Specific genes in this set included, but were not limited to, those genes associated with N assimilation (e.g., glutamate dehydrogenase, glutamine synthase, nitrate reductase), dissolved organic N utilization (e.g., urease, aminopeptidase, amino acid transport system), P scavenging (e.g., phosphate transporter, NPT), and DOP utilization (e.g., phosphatases) (Dataset 2). A number of these genes have been shown to be N-or P-responsive in transcriptional studies with cultures of the diatom T. pseudonana (56,61), and transporters and enzymes for the processing of organic N or P, as observed here, are well known to be RR in many phytoplankton (56,(62)(63)(64). Overall, these genes demonstrated patterns of regulation in situ (Fig.…”

Molecular insights into the niche of harmful brown tides

“…This would suggest that one strategy employed by A. anophagefferens during P deficiency is to produce more phosphate transporters, or switch to a more efficient one. This strategy has been observed in other eukaryotic algae (Chung et al, 2003;Dyhrman et al, 2006). Two tags (6248 and 1817) upregulated in the -P library mapped to two different 5′-nucleotidases.…”

“…For example, under N and P deficiency, some phytoplankton will induce genes for efficiently scavenging nutrients from a variety of sources and this induction can be seen at the transcriptional level (Grossman 2000, . Global transcriptome profiling studies have also examined nutrient deficiency responses in coccolithophores, dinoflagellates, diatoms, and the pelagophyte A. anophagefferens (Dyhrman et al 2006, Erdner and Anderson 2006, Mock et al 2008, Wurch et al 2011. A targeted study of N metabolism genes in A. anophagefferens demonstrated the up-regulation of transporters for nitrate, formate/nitrite, urea, ammonium, and amino acids among others during general N deficiency .…”

“…Phytoplankton have evolved mechanisms for efficiently scavenging N and P from a variety of sources and these mechanisms can be induced at the transcriptional level when a nutrient becomes limiting (Grossman, 2000;Dyhrman et al, 2006;. Global transcriptome profiling studies have shown broad transcriptional regulation to nutrient deficiency in coccolithophores and diatoms (Dyhrman et al, 2006;Mock et al, 2008). This has also been seen in the HAB-forming species Alexandrium fundyense (Erdner and Anderson, 2006), and transcriptional studies are an increasingly popular tool for studies of HAB nutritional physiology .…”

“…Long-SAGE tags are generated by the most 3′ Nla III restriction site on the transcript, and as a consequence, errors can be reduced by only considering tags mapping to the most 3′ Nla III site of a gene. Long-SAGE has been useful for identifying transcriptome profiles for other algae, including the coccolithophore Emiliania huxleyi (Dyhrman et al, 2006) and the dinoflagellate Pfiesteria shumwayae (Coyne et al, 2004).…”