Transcriptomic responses to grazing reveal the metabolic pathway leading to the biosynthesis of domoic acid and highlight different defense strategies in diatoms

Abstract

Background: A major cause of phytoplankton mortality is predation by zooplankton. Strategies to avoid grazers have probably played a major role in the evolution of phytoplankton and impacted bloom dynamics and trophic energy transport. Certain species of the genus Pseudo-nitzschia produce the neurotoxin, domoic acid (DA), as a response to the presence of copepod grazers, suggesting that DA is a defense compound. The biosynthesis of DA comprises fusion of two precursors, a C10 isoprenoid geranyl pyrophosphate and L-glutamate. Geranyl pyrophosphate (GPP) may derive from the mevalonate isoprenoid (MEV) pathway in the cytosol or from the methyl-erythritol phosphate (MEP) pathway in the plastid. L-glutamate is suggested to derive from the citric acid cycle. Fragilariopsis, a phylogenetically related but nontoxic genus of diatoms, does not appear to possess a similar defense mechanism. We acquired information on genes involved in biosynthesis, precursor pathways and regulatory functions for DA production in the toxigenic Pseudo-nitzschia seriata, as well as genes involved in responses to grazers to resolve common responses for defense strategies in diatoms.

Results: Several genes are expressed in cells of Pseudo-nitzschia when these are exposed to predator cues. No genes are expressed in Fragilariopsis when treated similarly, indicating that the two taxa have evolved different strategies to avoid predation. Genes involved in signal transduction indicate that Pseudo-nitzschia cells receive signals from copepods that transduce cascading molecular precursors leading to the formation of DA. Five out of seven genes in the MEP pathway for synthesis of GPP are upregulated, but none in the conventional MEV pathway. Five genes with known or suggested functions in later steps of DA formation are upregulated. We conclude that no gene regulation supports that L-glutamate derives from the citric acid cycle, and we suggest the proline metabolism to be a downstream precursor.

Conclusions: Pseudo-nitzschia cells, but not Fragilariopsis, receive and respond to copepod cues. The cellular route for the C10 isoprenoid product for biosynthesis of DA arises from the MEP metabolic pathway and we suggest proline metabolism to be a downstream precursor for L-glutamate. We suggest 13 genes with unknown function to be involved in diatom responses to grazers.

Keywords: Domoic acid; Fragilariopsis; Gene expression; Geranyl pyrophosphate; Grazer induced defense; L-Glutamate; Methyl-erythritol phosphate metabolic pathway; Proline; Pseudo-nitzschia.

Fig. 1

MA-plots for the gene expression data from Fragilariopsis and Pseudo-nitzschia. Both plots show the average expression of the normalized data on the x-axis and the log fold change ratios on the y-axis. Grey dots are non-differentially expressed genes (non-DE contigs) with a fold change below 1.5 and adjusted P-value above 0.05, number of replicates = 3. Blue dots are significantly differentially expressed genes (DE contigs) above the fold change threshold. Each dot represents a contig

Fig. 2

Breakdown of the major upregulated and downregulated genes in the KEGG and KOG database categories. The green box indicates candidate genes for the C10 isoprenoid in the biosynthesis of DA, number of replicates = 3

Fig. 3

Illustration of the major metabolic pathways for the biosynthesis of domoic acid (DA). The precursor of the C10 isoprenoid can either arise via the mevalonate (MEV) pathway that is located in the cytosol and/or an alternative route in the plastid, the methyl-erythritol phosphate metabolic (MEP) pathway. The tricarboxylic acid is proposedly derived from l-glutamate. The green arrow indicates where genes are upregulated when Pseudo-nitzschia is triggered to produce DA, the assigned enzymes are highlighted bold green, number of replicates = 3. Mean fold change (FC) and adjusted P values as well EC numbers for the enzymes and are given in Table 6