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 CO2 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.

FIG. 1.

Growth curves of E. huxleyi under replete, −N, and −P conditions. Graphs are plotted as cell number (filled symbols) and nutrient concentration (open symbols) versus day in culture, noting the difference in scale of the y axes. Error bars denote standard errors of the means (n = 4). Filled arrowhead indicates point of harvest.

FIG. 2.

(A) Differential expression of long SAGE tag 285. Frequency of tag 285 in the SAGE libraries generated from replete (control) and −N E. huxleyi (top). RT-PCR amplification of the fucoxanthin chlorophyll a/c binding protein gene that maps to tag 285 (115 bp) and type 1 actin (116 bp) from control and −N conditions (bottom). (B) Differential expression of long SAGE tag 45. Frequency of tag 45 in the SAGE libraries generated from control and −N E. huxleyi (top). RT-PCR amplification of the EST sequence which maps to tag 45 (267 bp) and type 1 actin (116 bp) from control and −N E. huxleyi (bottom). (C) Differential expression of long SAGE tag 12112. Frequency of tag 12112 in the SAGE libraries generated from control and −P E. huxleyi (top). RT-PCR amplification of a 286-bp fragment of the putative inorganic pyrophosphatase gene that maps to tag 12112 and a 116-bp fragment of type 1 actin from control and −P samples (bottom). All PCRs were performed with equal template concentrations.