Evidence for a role of gibberellins in salicylic acid-modulated early plant responses to abiotic stress in Arabidopsis seeds

Abstract

Exogenous application of gibberellic acid (GA(3)) was able to reverse the inhibitory effect of salt, oxidative, and heat stresses in the germination and seedling establishment of Arabidopsis (Arabidopsis thaliana), this effect being accompanied by an increase in salicylic acid (SA) levels, a hormone that in recent years has been implicated in plant responses to abiotic stress. Furthermore, this treatment induced an increase in the expression levels of the isochorismate synthase1 and nonexpressor of PR1 genes, involved in SA biosynthesis and action, respectively. In addition, we proved that transgenic plants overexpressing a gibberellin (GA)-responsive gene from beechnut (Fagus sylvatica), coding for a member of the GA(3) stimulated in Arabidopsis (GASA) family (FsGASA4), showed a reduced GA dependence for growth and improved responses to salt, oxidative, and heat stress at the level of seed germination and seedling establishment. In 35S:FsGASA4 seeds, the improved behavior under abiotic stress was accompanied by an increase in SA endogenous levels. All these data taken together suggest that this GA-responsive gene and exogenous addition of GAs are able to counteract the inhibitory effects of these adverse environmental conditions in seed germination and seedling growth through modulation of SA biosynthesis. Furthermore, this hypothesis is supported by the fact that sid2 mutants, impaired in SA biosynthesis, are more sensitive to salt stress than wild type and are not affected by exogenous application of GA(3).

Figure 1.

Northern-blot analysis of total RNA isolated from beechnut seeds imbibed at 4°C in 100 mm ABA, water, and 100 mm GA₃ from 1 to 6 weeks. Ten micrograms of RNA were used per lane and hybridized with a FsGASA4 cDNA probe. Top section: Ethidium bromide-stained gel showing RNAs. The numbers indicate weeks of imbibition.

Figure 2.

Molecular analysis of Arabidopsis wild-type (Col-0) and 35S:FsGASA4 transgenic lines (G1–G3). A, RNA-blot analysis. Total RNA (10 μg) was isolated and hybridized with a specific FsGASA4 probe. Bottom: Ethidium bromide-stained gel showing rRNAs. B, Southern-blot analysis. Genomic DNA was digested with HindIII, blotted onto a nylon membrane, and hybridized with a FsGASA4-specific probe.

Figure 3.

Effect of PCB on plant growth. Plant phenotypes after 60 d growth in 10 μm PCB (Col-0, wild-type seeds; G1–G5, FsGASA4 transgenic lines) are shown.

Figure 4.

Percentages of Col-0 seeds that completed germination and developed fully expanded green cotyledons after 10 d under: 150 mm NaCl (A), 0.5 mm paraquat (B), heat treatment (50°C; C), 50 μm GA₃ plus 150 mm NaCl (D), 50 μm GA₃ plus 0.5 mm paraquat (E), and heat treatment (50°C; F) in the presence of 50 μm GA₃. G, Differences in heat tolerance (50°C) in wild-type and transgenic lines treated or not with 50 μm GA₃. Approximately 100 seeds of each line were sowed and scored. Data are means ± sd of three independent experiments. Asterisks denote significant differences at P ≤ 0.05 between treated and nontreated seeds.

Figure 5.

Percentages of Col-0 seeds (treated or not with SA) that completed germination and developed fully expanded green cotyledons after 10 d under: 150 mm NaCl (A), 0.5 mm paraquat (B), and heat treatment (50°C). Approximately 100 seeds of each line were sowed and scored. Data are means ± sd of three independent experiments. Asterisks denote significant differences at P ≤ 0.05 between treated and nontreated seeds.

Figure 6.

Expression of the ics1, npr1, and RGA in FsGASA-overexpressing plants (G1–G3) compared to Col-0. mRNA levels of the indicated genes were determined by northern-blot analysis using total RNAs (10 μg/line) isolated from 7-d-old seedlings. Bottom: Ethidium bromide-stained gel showing rRNAs. Top section: Quantification of hybridization signals obtained by using a phosphoimage scanner. Data were normalized to the rRNA value. Blots were repeated twice and yielded similar results.

Figure 7.

MDA content in wild-type and G1 to G4 FsGASA4 transgenic lines under normal conditions (C1), and after heat stress at 50°C for 3 h (C2) treated with 50 mm GA₃ and 50 mm SA.

Figure 8.

Expression of the ics1 and npr1 genes in Arabidopsis seedlings. Total RNA was isolated from 7-d-old seedlings, treated or not with 100 μm GA₃. Bottom: Ethidium bromide-stained gel showing rRNAs. Top section: Quantification of hybridization signals obtained by using a phosphoimage scanner. Data were normalized to the rRNA value. Blots were repeated twice and yielded similar results.

Figure 9.

Effect of SA on ga1 mutant seed germination and seedling growth. A, Percentages of ga1 seeds that completed germination and developed green cotyledons after 15 d in the presence or not of 50 μm SA. B, Phenotypes of ga1 seeds and seedlings after 15 and 40 d in the presence or not of 50 μm SA. Data are means ± sd of three independent experiments.