Transcriptional and metabolomic analysis of Ascophyllum nodosum mediated freezing tolerance in Arabidopsis thaliana

Abstract

Background: We have previously shown that lipophilic components (LPC) of the brown seaweed Ascophyllum nodosum (ANE) improved freezing tolerance in Arabidopsis thaliana. However, the mechanism(s) of this induced freezing stress tolerance is largely unknown. Here, we investigated LPC induced changes in the transcriptome and metabolome of A. thaliana undergoing freezing stress.

Results: Gene expression studies revealed that the accumulation of proline was mediated by an increase in the expression of the proline synthesis genes P5CS1 and P5CS2 and a marginal reduction in the expression of the proline dehydrogenase (ProDH) gene. Moreover, LPC application significantly increased the concentration of total soluble sugars in the cytosol in response to freezing stress. Arabidopsis sfr4 mutant plants, defective in the accumulation of free sugars, treated with LPC, exhibited freezing sensitivity similar to that of untreated controls. The 1H NMR metabolite profile of LPC-treated Arabidopsis plants exposed to freezing stress revealed a spectrum dominated by chemical shifts (δ) representing soluble sugars, sugar alcohols, organic acids and lipophilic components like fatty acids, as compared to control plants. Additionally, 2D NMR spectra suggested an increase in the degree of unsaturation of fatty acids in LPC treated plants under freezing stress. These results were supported by global transcriptome analysis. Transcriptome analysis revealed that LPC treatment altered the expression of 1113 genes (5%) in comparison with untreated plants. A total of 463 genes (2%) were up regulated while 650 genes (3%) were down regulated.

Conclusion: Taken together, the results of the experiments presented in this paper provide evidence to support LPC mediated freezing tolerance enhancement through a combination of the priming of plants for the increased accumulation of osmoprotectants and alteration of cellular fatty acid composition.

Figure 1

Proline accumulation and expression of proline metabolism genes in wild-type (WT) Arabidopsis: (A) effect of ANE and LPC on the accumulation of proline in the leaves of wild type Arabidopsis (Col-0) in response to freezing stress (Data are the means ± SE) and (B) Real time-PCR analysis of expression of genes encoding Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and prolinedehydogenase (ProDH). Bar with * is significantly different (P ≤ 0.05) from that of the control treatment.

Figure 2

Ascophyllum nodosum extracts did not rescue the freezing sensitive phenotype of p5cs1 mutants: (A) Petri Plate Freezing Tolerance Assay with wild-type Arabidopsis (left partition of Petri plate) and p5cs1 mutants (right partition of Petri plate) treated with (i) Control (ii) Ascophyllum nodosum extract (ANE) and (iii) lipophilic component of ANE (LPC), (B) survival rate at different freezing temperatures of wild type Arabidopsis and p5cs1 mutant plants treated with ANE or LPC in Petri Plates, (C) proline accumulation of p5cs1 mutants treated with ANE or LPC in response to freezing in peat pellets, and (D) Phenotypic responses of p5cs1 mutant plants treated with (i) water control (ii) ANE (1.0 g L^-1) and (iii) LPC (1.0 g L^-1) to a temperature of −2°C for 24 h in Peat pellet freezing assay. Bars are the means ± SE.

Figure 3

Accumulation of soluble sugars in wild-type Arabidopsis plants treated with ANE or LPC under different freezing and thawing regimes. Data are the means ± SE, Bar with * is significantly different (P ≤ 0.05) from that of the control treatment.

Figure 4

Sugar accumulation is required for ANE-mediated freezing tolerance in Arabidopsis: (A) sensitive phenotype of sfr4 mutant plants treated with (i) water control (ii) ANE (1.0 g L^-1) and (iii) LPC (1.0 g L^-1) to a temperature of −2.5°C for 24 h in Peat Pellet Freezing Tolerance Assay, and (B) extent of freezing-induced tissue damage in controls and treated sfr4 mutant plants as revealed by trypan blue staining; comparison of the area of tissue damage in the trypan blue stained leaves using the image processing and analysis software Image-j®. Data are means ± SE.

Figure 5

¹H-NMR metabolite profile spectrum of wild-type Arabidopsis plants treated with LPC during freezing temperature of −2°C for 24 h.

Figure 6

2D NMR spectra of metabolites extracted from LPC-treated plants, upon exposure to freezing stress of −2.5°C for 24 h: (A) 2D NMR TOCSY spectrum showing characteristic proton correlations in unsaturated fatty acids and (B) 2D NMR HSQC spectrum showing characteristic proton and carbon signals of unsaturated fatty acids and sugars or sugar alcohols.

Figure 7

Diagram of representative metabolic pathways regulated by the LPC treatment during freezing stress. Each pathway is shown as a glyph consisting of nodes and lines, which represent the metabolites and reactions, respectively. Expression-level change of each reaction is shown in a color relative to the expression level, as indicated in the color scale bar. Triangle, amino acids; square, carbohydrates; diamond, proteins; open circle, others; closed circle, phosphorylated.

Figure 8

Venn diagram showing the number of significantly (a) up or (b) down-regulated and the shared genes during freezing stress and post-freezing recovery period.