Priming with a Seaweed Extract Strongly Improves Drought Tolerance in Arabidopsis

Abstract

Drought represents a major threat to plants in natural ecosystems and agricultural settings. The biostimulant Super Fifty (SF), produced from the brown alga Ascophyllum nodosum, enables ecologically friendly stress mitigation. We investigated the physiological and whole-genome transcriptome responses of Arabidopsis thaliana to drought stress after a treatment with SF. SF strongly decreased drought-induced damage. Accumulation of reactive oxygen species (ROS), which typically stifle plant growth during drought, was reduced in SF-primed plants. Relative water content remained high in SF-treated plants, whilst ion leakage, a measure of cell damage, was reduced compared to controls. Plant growth requires a functional shoot apical meristem (SAM). Expression of a stress-responsive negative growth regulator, RESPONSIVE TO DESICCATION 26 (RD26), was repressed by SF treatment at the SAM, consistent with the model that SF priming maintains the function of the SAM during drought stress. Accordingly, expression of the cell cycle marker gene HISTONE H4 (HIS4) was maintained at the SAMs of SF-primed plants, revealing active cell cycle progression after SF priming during drought. In accordance with this, CYCP2;1, which promotes meristem cell division, was repressed by drought but enhanced by SF. SF also positively affected stomatal behavior to support the tolerance to drought stress. Collectively, our data show that SF priming mitigates multiple cellular processes that otherwise impair plant growth under drought stress, thereby providing a knowledge basis for future research on crops.

Keywords: Ascophyllum nodosum; abiotic stress; drought; priming; reactive oxygen species.

Figure 1

Response of Super Fifty (SF)-primed and unprimed plants to drought stress. (a) Arabidopsis plants after 11 days of drought stress, at 42 days after germination (DAG). In the absence of SF priming, drought-stressed plants (H₂O+drought stress (Dr)) show stress-related symptoms (wilting of most leaves), while drought stress-related symptoms are seemingly absent in SF-primed plants (SF+Dr). Leaves no. 8, 9, and 10, harvested for ion conductivity and relative water content (RWC) measurements—see panels (d,e)—are indicated by white arrows (representative images). (b) Rosette diameter. (c) Number of rosette leaves. Data in (b,c) were obtained for plants at 40 DAG. (d) Ion conductivity in leaves as a measure of membrane damage. (e) Relative water content in leaves. Error bars denote the standard deviation of the mean (SD). Treatments sharing same letters indicate nonsignificant differences, while treatments with different letters indicate significant differences (p < 0.05; one-way ANOVA, Tukey’s multiple comparison test). Dr, drought stress; SF, Super Fifty.

Figure 2

SF priming triggers a reduction in reactive oxygen species (ROS) levels. (a) Histochemical staining of ROS in leaves using 3,3′-diaminobenzidine (DAB). (b) Hydrogen peroxide levels in leaves of 15-day-old Arabidopsis Col-0 plants without (H₂O+H₂O) and with SF treatment (SF+H₂O) in the absence of drought stress. (c) Hydrogen peroxide levels in leaves at 42 DAG. Note the higher H₂O₂ levels in unprimed plants (H₂O+Dr) compared to SF-primed plants (SF+Dr) under drought stress. Quantification of H₂O₂ levels (b,c) was performed using the Amplex Red Hydrogen Peroxide/Peroxidase kit as reported [30]. Error bars in (b,c) denote the standard deviation of the mean (SD). In (b), asterisks (**) indicate significant difference (n = 4) (p < 0.01, Student’s t-test). In (c), different letters indicate statistically significant differences between the samples (p < 0.05, n = 8; one-way ANOVA, Tukey’s multiple comparison test). FW, fresh weight; Dr, drought stress; SF, Super Fifty.

Figure 3

SF priming-induced transcript changes during drought stress in Arabidopsis. (a) Number of differentially expressed genes (DEGs) in stressed (H₂O+Dr) vs. unstressed (H₂O+H₂O), and in primed (SF+Dr) vs. unprimed (H₂O+Dr) plants during drought. The list of DEGs is provided in Supplementary File S1. (b) Gene ontology (GO) enrichment of DEGs in SF-primed and stressed (SF+Dr) plants categorized into different groups based on the biological process. The RichFactor represents the ratio of the number of DEGs annotated with the given GO term to the number of all genes annotated with the respective GO term. The sizes and colours of the dots represent the number of genes and FDR value, respectively. The list of enriched GO terms is provided in Supplementary File S2. Dr, drought stress; SF, Super Fifty.

Figure 4

Ascorbate peroxidase activity in Arabidopsis leaves. Error bars denote the standard deviation (SD) of the mean. Different letters represent significant differences between the treatment means (p < 0.05, n = 3; one-way ANOVA, Tukey’s multiple comparison test). Dr, drought stress; SF, Super Fifty.

Figure 5

Expression of RD26 and HIS4 in the shoot apical meristems (SAMs) of SF-primed Arabidopsis plants. Expression of both genes was determined by RNA in situ hybridization. (a) Drought-induced expression of RD26 in the SAMs of SF-primed and unprimed plants. Note the moderate expression of RD26 in unprimed, drought-stressed plants at 36 DAG (days after germination; day 5 of drought) and the highly induced expression at 42 DAG (11 days of drought). (b) Expression of HIS4 (marker of the S phase of the cell cycle) in the SAM. (c) Number of HIS4-positive cells in the SAMs of SF-primed and unprimed plants. In (b,c), note the absence (NA) of HIS4-positive cells in unprimed drought-stressed plants at 42 DAG (i.e., after 11 days of drought), but the high number of HIS4-positive cells in SAMs of SF-primed plants, even after extended drought. Error bars indicate standard deviation (n = 3). Statistical significance was calculated using Student’s t-test (* p < 0.05; ** p < 0.01). Dr, drought stress; SF, Super Fifty.

Figure 6

Stomatal closure response under drought. (a) Stomatal apertures in SF-primed and unprimed plants with the progression of drought stress. Note that, under well-watered conditions, SF priming (SF+H₂O) leads to partial stomatal closure compared to nonprimed plants (H₂O+H₂O). Under conditions of drought stress, partial stomatal closure occurred earlier in SF-primed plants (SF+Dr) than in nonprimed plants (H₂O+Dr). Error bars indicate standard error of the mean (SEM), n ≥ 250; treatments sharing different letters indicate significant differences from each other (p < 0.05; one-way ANOVA, Tukey’s multiple comparison test). (b) Relative expression of PRX34 in SF-primed and unprimed plants. Values are expressed as fold change (FC) in expression normalized to those determined in unprimed, well-watered (H₂O+H₂O) controls. Asterisks represent significant differences in expression in unprimed drought-stressed (H₂O+Dr) vs. primed (SF+Dr and SF+H₂O) plants (p < 0.0001; one-way ANOVA, Tukey’s multiple comparison test). (c) Histochemical staining assay for ROS accumulation in leaf stomata. Leaf epidermal peels from plants (unprimed and primed) under drought stress were used for DAB staining following a reported protocol [74]. Red arrow heads indicate stomata. Dr, drought stress; SF, Super Fifty.

Figure 7

Response to drought in arr2-5 and prx34-2 mutants. (a) Response of plants exposed to 11 days of drought. (b) Ion conductivity as a measure of membrane damage. (c) Relative water content. Data are shown for wild type (Col-0), arr2-5 and prx34-2 plants (leaves 10 and 11). Treatments sharing different letters represent statistically significant differences from each other (p < 0.05; one-way ANOVA, Tukey’s multiple comparisons test). Dr, drought stress; SF, Super Fifty.