Global transcriptional profiling of the toxic dinoflagellate Alexandrium fundyense using Massively Parallel Signature Sequencing

Abstract

Background: Dinoflagellates are one of the most important classes of marine and freshwater algae, notable both for their functional diversity and ecological significance. They occur naturally as free-living cells, as endosymbionts of marine invertebrates and are well known for their involvement in "red tides". Dinoflagellates are also notable for their unusual genome content and structure, which suggests that the organization and regulation of dinoflagellate genes may be very different from that of most eukaryotes. To investigate the content and regulation of the dinoflagellate genome, we performed a global analysis of the transcriptome of the toxic dinoflagellate Alexandrium fundyense under nitrate- and phosphate-limited conditions using Massively Parallel Signature Sequencing (MPSS).

Results: Data from the two MPSS libraries showed that the number of unique signatures found in A. fundyense cells is similar to that of humans and Arabidopsis thaliana, two eukaryotes that have been extensively analyzed using this method. The general distribution, abundance and expression patterns of the A. fundyense signatures were also quite similar to other eukaryotes, and at least 10% of the A. fundyense signatures were differentially expressed between the two conditions. RACE amplification and sequencing of a subset of signatures showed that multiple signatures arose from sequence variants of a single gene. Single signatures also mapped to different sequence variants of the same gene.

Conclusion: The MPSS data presented here provide a quantitative view of the transcriptome and its regulation in these unusual single-celled eukaryotes. The observed signature abundance and distribution in Alexandrium is similar to that of other eukaryotes that have been analyzed using MPSS. Results of signature mapping via RACE indicate that many signatures result from sequence variants of individual genes. These data add to the growing body of evidence for widespread gene duplication in dinoflagellates, which would contribute to the transcriptional complexity of these organisms. The MPSS data also demonstrate that a significant number of dinoflagellate mRNAs are transcriptionally regulated, indicating that dinoflagellates commonly employ transcriptional gene regulation along with the post-transcriptional regulation that has been well documented in these organisms.

Figure 1

Growth, nutrient and toxin characteristics of nutrient-limited cultures. Cell densities were recorded daily in nitrate- (A) and phosphate- (B) limited cultures of A. fundyense. Arrows indicate the day that samples were collected for MPSS analysis. After harvest, the cultures were enriched with the limiting nutrient and changes in cell number were monitored. Nutrient concentrations (open symbols) and toxin per cell (closed symbols) were also monitored daily in the nitrate- (C) and phosphate- (D) limited cultures.

Figure 2

Signature abundance distributions. The abundance of each signature is given as a percentage of total signatures in the N/40 (grey bars) and P/40 (black bars) libraries. For example, 1% abundance indicates a signature that is present 10,000 times in each 1,000,000 sequences (10,000 tpm).

Figure 3

Comparison of expression for all of the A. fundyense signatures. The expression ratios compare the abundance of each signature between the two libraries. Bars indicate the number of signatures with an expression ratio within the stated range. Expression ratios were calculated to be ≥1, so that signatures that are more highly expressed in the P/40 library are to the left (P:N) whereas those with greater expression in the N/40 library are to the right (N:P). The exceptions are signatures with ratio = 1, these are repeated on the two sides of the figure, and those with ratio = 0.

Figure 4

Paired abundance data for all signatures with differential expression significant at p < 0.05. Each data point corresponds to a signature with expression that is significantly different between the N/40 and P/40 libraries at p < 0.05. Data are plotted as signature expression in the P/40 library (x-axis) vs. expression in the N/40 library (y-axis). The axis scale decreases from left to right panels to show detail. Dashed line is the line of constant expression, i.e. the point at which the expression ratio is two-fold different between the two conditions.