Transcriptomic and metabolomic analysis of copper stress acclimation in Ectocarpus siliculosus highlights signaling and tolerance mechanisms in brown algae

Abstract

Background: Brown algae are sessile macro-organisms of great ecological relevance in coastal ecosystems. They evolved independently from land plants and other multicellular lineages, and therefore hold several original ontogenic and metabolic features. Most brown algae grow along the coastal zone where they face frequent environmental changes, including exposure to toxic levels of heavy metals such as copper (Cu).

Results: We carried out large-scale transcriptomic and metabolomic analyses to decipher the short-term acclimation of the brown algal model E. siliculosus to Cu stress, and compared these data to results known for other abiotic stressors. This comparison demonstrates that Cu induces oxidative stress in E. siliculosus as illustrated by the transcriptomic overlap between Cu and H2O2 treatments. The common response to Cu and H2O2 consisted in the activation of the oxylipin and the repression of inositol signaling pathways, together with the regulation of genes coding for several transcription-associated proteins. Concomitantly, Cu stress specifically activated a set of genes coding for orthologs of ABC transporters, a P1B-type ATPase, ROS detoxification systems such as a vanadium-dependent bromoperoxidase, and induced an increase of free fatty acid contents. Finally we observed, as a common abiotic stress mechanism, the activation of autophagic processes on one hand and the repression of genes involved in nitrogen assimilation on the other hand.

Conclusions: Comparisons with data from green plants indicate that some processes involved in Cu and oxidative stress response are conserved across these two distant lineages. At the same time the high number of yet uncharacterized brown alga-specific genes induced in response to copper stress underlines the potential to discover new components and molecular interactions unique to these organisms. Of particular interest for future research is the potential cross-talk between reactive oxygen species (ROS)-, myo-inositol-, and oxylipin signaling.

Figure 1

Physiological effects of acute copper (Cu) toxicity on E. siliculosus strain Ec32. Changes in the photosynthetic yield (Fv/Fm) were monitored during 8 h in absence of Cu (diamonds), and in presence of 250 (squares) and 500 μg L^-1 (triangles) of CuCl₂ (final concentration). Values represent means of three independent replicates and bars represent the standard error. Asterisks highlight significantly different values from the respective control condition (U-test, p < 0.05).

Figure 2

Functional distribution of contigs/singletons up-regulated and down-regulated in Cu-stressed E. siliculosus. Contigs/singletons were manually classified according to their annotation in the E. siliculosus genome database. The figure shows the number of significantly down- (left) and up-regulated (right) contig/singletons in each category (two-way ANOVA, FDR < 5%).

Figure 3

Phylogenetic tree of human, A. thaliana, and E. siliculosus (red and blue) ABC transporters.E. siliculosus genes induced specifically by Cu stress are marked in red. Confidence values are the results of an approximate likelihood ratio test; only confidence values ≥ 50 are shown.

Figure 4

Metabolic pathways related to primary metabolism altered under short-term copper stress identified by transcriptomic analysis. HSPs, heat shock proteins; LHC, light harvesting complex.

Figure 5

Heat map of compounds identified by UPLC-MS and GC-MS in copper stress and control conditions. Samples were arranged according to a hierarchical clustering analysis (Euclidean distance), and the 47 compounds identified were grouped manually. Concentrations of each metabolite were normalized to a maximum of 1 (see Methods section). “*” and “†” indicate significant results (FDR < 5%) in the two-way ANOVA for stress and the interaction term “stress* time”, respectively. 9-HpOTrE, 9-hydroperoxy-10,12,15-octadecatrienoic acid; 13-HpOTrE, 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid; HpOTrE 3; oxylipin with the same m/z and raw formula as 11- or 15- hydroperoxy-9Z,11E,15Z-octadecatrienoic acid; 13-HpODE, 13-hydroperoxy-9Z,11E-octadecadienoic acid; 13-HOTrE, 13S-hydroxy-9Z,11E,15Z-octadecatrienoic acid; 13-HODE, 13-hydroxy-9Z,11E-octadecadienoic acid; 12-oxo-PDA, 12-oxophytodienoic acid; MeJA, methyl-jasmonate; PGJ2, prostaglandin J2; PGA2, prostaglandin A2, PGB2, prostaglandin B2; oxo-ETE, oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid; LTB4, leukotriene B4; GABA, γ-aminobutyric acid.

Figure 6

Changes in contents of free polyunsaturated fatty acids and oxylipins under copper stress. 9-HpOTrE, 9-hydroperoxy-10,12,15-octadecatrienoic acid; 13-HpOTrE, 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid; 13-HpODE, 13-hydroperoxy-9Z,11E-octadecadienoic acid; 13-HOTrE, 13S-hydroxy-9Z,11E,15Z-octadecatrienoic acid; 13-HODE, 13-hydroxy-9Z,11E-octadecadienoic acid; 12-oxo-PDA, 12-oxophytodienoic acid; PGJ2, prostaglandin J2; PGA2, prostaglandin A2; PGB2, prostaglandin B2; oxo-ETE, oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid; LTB4, leukotriene B4.