An Ascophyllum nodosum-Derived Biostimulant Protects Model and Crop Plants from Oxidative Stress

Abstract

Abiotic stresses, which at the molecular level leads to oxidative damage, are major determinants of crop yield loss worldwide. Therefore, considerable efforts are directed towards developing strategies for their limitation and mitigation. Here the superoxide-inducing agent paraquat (PQ) was used to induce oxidative stress in the model species Arabidopsis thaliana and the crops tomato and pepper. Pre-treatment with the biostimulant SuperFifty (SF) effectively and universally suppressed PQ-induced leaf lesions, H₂O₂ build up, cell destruction and photosynthesis inhibition. To further investigate the stress responses and SF-induced protection at the molecular level, we investigated the metabolites by GC-MS metabolomics. PQ induced specific metabolic changes such as accumulation of free amino acids (AA) and stress metabolites. These changes were fully prevented by the SF pre-treatment. Moreover, the metabolic changes of the specific groups were tightly correlating with their phenotypic characteristics. Overall, this study presents physiological and metabolomics data which shows that SF protects against oxidative stress in all three plant species.

Keywords: Ascophyllum nodosum; biostimulant; oxidative stress.

Figure 1

SuperFifty (SF) protects leaves from damage, caused by oxidative stress. Plants on the left are treated with paraquat (PQ), a herbicide that elevates the endogenous levels of ROS and triggers cell death (15 µM for Arabidopsis and 25 µM for tomato and pepper), and have clearly visible lesions. Plants on the right were pre-treated with 1% aqueous solution of SF, followed by the same treatment with PQ. Control plants, sprayed only with water (H₂O) or only with 1% SF, show no signs of effect (pictures not shown).

Figure 2

Damage reduction, caused by SF pre-treatment. (A) SF reduces leaf lesion area in PQ-treated plants. The dead leaf surface, as a parameter indicating cell damage, of the four variants (as described in Materials and Methods) of Arabidopsis, pepper, and tomato (green, blue and yellow color bars, respectively) was assessed with the ImageJ software. n = 12 for Arabidopsis, n = 48 for pepper and tomato. (B) SF prevents excess ion leakage in PQ-treated plants. Measurement of electrolyte leakage in leaves of Arabidopsis, pepper and tomato exposed to four different treatments, relative to (−SF/−PQ) control. n = 36 for Arabidopsis, n = 48 for pepper and tomato. Data are means ±SEM (standard error of the mean) of three biological replicates. Differences between two single treatments were tested by Welch’s t-test, with p < 0.05. Statistical significance relative to controls from the same species is indicated with asterisks as follows: p ≤ 0.0001 (****), p ≤ 0.001 (***), p ≤ 0.01 (**), p ≤ 0.05 (*).

Figure 3

SF protects from accumulation of H₂O₂ in PQ treated plants. (A) DAB-stained leaves of Arabidopsis, pepper and tomato exposed to different treatments (B) The percentage of DAB-stained area of the four variants of Arabidopsis, tomato, and pepper was assessed with the ImageJ software as described in Materials and Methods (n = 6). Data are means ±SEM of three biological replicates. Differences between two single treatments were tested by Welch’s t-test, with p < 0.05. Statistical significance relative to controls from the same species is indicated with asterisks as follows: p ≤ 0.0001 (****), p ≤ 0.001 (***), p ≤ 0.01 (**), p ≤ 0.05 (*).

Figure 4

SF pretreatment alleviates the reduction of chlorophyll fluorescence parameters induced by oxidative stress. The maximum quantum yield (A) and fluorescence decrease ratio (B) in dark-adapted Arabidopsis, pepper and tomato plants (n = 12), treated or not with SF and PQ, were measured by PAM fluorometry. Data are means ±SEM of three biological replicates. Differences between two single treatments were tested by Welch’s t-test, with p < 0.05. Statistical significance relative to controls from the same species is indicated in the Table as follows: p ≤ 0.0001 (****), p ≤ 0.001 (***), p ≤ 0.01 (**), ns = not significant.

Figure 5

2D sPLSDA Scores plots for 89 metabolites of Arabidopsis, pepper and tomato. The −SF/+PQ treatment (light green horizontally arranged clusters) always groups separately from the others, which showing much more similar distribution and partial or complete overlapping (all others, generally vertical clusters) (n = 6). AT—Arabidopsis thaliana, CA—Capsicum annuum, SL—Solanum lycopersicum.

Figure 6

Scatter plots of plant phenotype parameters versus sPLSDA component 1 values. Mean sPLSDA component 1 values were compared between treatment groups using one-way ANOVA, with Tukey’s multiple comparisons test applied to correct for multiple testing. For all three plant species, a significant difference was observed between the −SF/+PQ (oxidatively stressed) and all other treatment groups. This was observed for both the sPLSDA component 1 values and for all plant phenotypes measured. Horizontal error bars denote the standard error of the mean (SEM) of sPLSDA component 1 values. Vertical error bars denote the SEM of phenotype parameters. Asterisks denote statistically significant differences between treatment groups and the untreated control (−SF/−PQ) as follows: p ≤ 0.0001 (****), p ≤ 0.001 (***), p ≤ 0.01 (**), p ≤ 0.05 (*). Horizontal and vertical asterisks denote significant differences between treatment groups and the untreated control for phenotype parameters and sPLSDA component 1 values respectively. Full data are given in Supplementary Materials (Supplementary Figures S4 and S5) and Supplementary Table S1.

Figure 7

Levels of primary metabolites, relative to the negative control (−SF/−PQ) for each of the species, clustered for similarity of distribution patterns. The length of the lateral arms of the dendrograms represent the degree of resemblance. Significance can be seen in Supplementary Table S2 (n = 6). AT—Arabidopsis thaliana, CA—Capsicum annuum, SL—Solanum lycopersicum.