Salt Induces Features of a Dormancy-Like State in Seeds of Eutrema (Thellungiella) salsugineum, a Halophytic Relative of Arabidopsis

Abstract

The salinization of land is a major factor limiting crop production worldwide. Halophytes adapted to high levels of salinity are likely to possess useful genes for improving crop tolerance to salt stress. In addition, halophytes could provide a food source on marginal lands. However, despite halophytes being salt-tolerant plants, the seeds of several halophytic species will not germinate on saline soils. Yet, little is understood regarding biochemical and gene expression changes underlying salt-mediated inhibition of halophyte seed germination. We have used the halophytic Arabidopsis relative model system, Eutrema (Thellungiella) salsugineum to explore salt-mediated inhibition of germination. We show that E. salsugineum seed germination is inhibited by salt to a far greater extent than in Arabidopsis, and that this inhibition is in response to the osmotic component of salt exposure. E. salsugineum seeds remain viable even when germination is completely inhibited, and germination resumes once seeds are transferred to non-saline conditions. Moreover, removal of the seed coat from salt-treated seeds allows embryos to germinate on salt-containing medium. Mobilization of seed storage reserves is restricted in salt-treated seeds, while many germination-associated metabolic changes are arrested or progress to a lower extent. Salt-exposed seeds are further characterized by a reduced GA/ABA ratio and increased expression of the germination repressor genes, RGL2, ABI5, and DOG1. Furthermore, a salt-mediated increase in expression of a LATE EMBRYOGENESIS ABUNDANT gene and accretion of metabolites involved in osmoprotection indicates induction of processes associated with stress tolerance, and accumulation of easily mobilized carbon reserves. Overall, our results suggest that salt inhibits E. salsugineum seed germination by inducing a seed state with molecular features of dormancy while a physical constraint to radicle emergence is provided by the seed coat layers. This seed state could facilitate survival on saline soils until a rain event(s) increases soil water potential indicating favorable conditions for seed germination and establishment of salt-tolerant E. salsugineum seedlings.

Keywords: Arabidopsis relative; Brassicaceae; Eutrema salsugineum; dormancy; extremophile plants; halophyte; salt stress; seed germination.

FIGURE 1

Germination of Arabidopsis thaliana and Eutrema salsugineum seeds under different stresses. Seeds were sown on plates containing half-strength MS medium with or without the indicated concentrations of each treatment (mM). (A,B) NaCl. (C,D) Mannitol. (E,F) LiCl. Germination was recorded at the indicated time points and expressed as a percentage of the total number of seeds on the plate. Data are mean ± SD (n = 3). Each replicate plate contained ca. 100 seeds. For statistical analysis, see Supplementary Table S2. Data are representative of two independent experiments. N.B. In panels (C) and (E), the control treatment line is not visible as it overlaps with the 100 and 200 mM NaCl or 5 and 10 mM LiCl treatments, respectively.

FIGURE 2

Eutrema salsugineum seeds remain viable after NaCl treatment. (A,B) 2,3,5-triphenyltetrazolium chloride was used to stain for seed viability. (A) NaCl-treated seeds at 48 h after stratification. (B) Autoclaved seeds used as a negative control. (C) Salt-inhibited E. salsugineum seeds germinate when transferred to non-saline conditions. Seeds were sown on plates containing half-strength MS medium either without or supplemented with 200 mM NaCl. At 2 days after stratification (time point indicated by arrow), non-germinated seeds from salt-containing plates were rinsed with water and rescued to 0 mM NaCl plates. Germination was recorded daily and expressed as a percentage of the total number of seeds on the plate. Data are mean ± SD (n = 4). Each replicate plate contained ca. 100 seeds. Data are representative of two independent experiments. Dark gray line, control; light gray line, 200 mM NaCl.

FIGURE 3

Changes in protein and lipid content in germinating E. salsugineum seeds. Coomassie blue-stained SDS-PAGE gel of albumin (A,B) and globulin (C,D) fractions of seed storage proteins. (E) Changes in total seed fatty acid (TFA) content. Data are mean ± SD (n = 3). Data are representative of two independent experiments. Bars with different letters indicate significant difference at P < 0.05 (Student’s t-test). Dark gray bars, control; light gray bars, 200 mM NaCl; M, Size marker (kDa); D, Dry seeds; 0–48, hours after stratification.

FIGURE 4

Principal component analysis (PCA) of metabolic profiles of control and NaCl-treated E. salsugineum seeds. Variance explained by each component is indicated in brackets. The first principal component (PC1) separates samples by time after stratification specifically for seeds under control conditions. PC2 discriminates between control and salt-treated samples particularly at the early (0 HAS) and late stages (36 and 48 HAS) of germination. Data are representative of similar results from two independent experiments. HAS, hours after stratification.

FIGURE 5

Effect of salt on changes in primary metabolism in germinating E. salsugineum seeds. (A) Heat map of relative metabolite content in control and NaCl-treated E. salsugineum seeds. Data are expressed as log₁₀ transformed values. Only metabolites with significantly different levels of abundance (P < 0.05) according to the two-way ANOVA analysis (Supplementary Table S5) are shown. Data are representative of similar results from two independent experiments. (B) Relative abundance of selected metabolites in germinating E. salsugineum seeds. Data are mean ± SD (n = 5) and are representative of two independent experiments. Asterisks represent significant difference (P < 0.05, Student’s t-test, Bonferroni correction) between salt-treated and control seeds at each time point. Dark gray bars, Control; light gray bars, 200 mM NaCl; D, Dry seeds; 0–48, hours after stratification.

FIGURE 6

Effect of salt on phytohormone levels in germinating E. salsugineum seeds. (A) Abscisic acid (ABA). (B) Gibberellic acid (GA4). (C) Auxin (IAA). (D) Jasmonic acid (JA). Data are mean ± SD (n = 3). Bars with different letters indicate significant difference at P < 0.05 (Student’s t-test). Dark gray bars, Control; light gray bars, 200 mM NaCl; D, Dry seeds, 0–48, hours after stratification.

FIGURE 7

Effect of salt on expression of genes related to hormone levels, signaling, and dormancy in germinating E. salsugineum seeds. (A) NCED6. (B) CYP707A1. (C) CYP707A3. (D) RGL2. (E) ABI5. (F) EM6. (G) DOG1. Gene expression was determined by real-time qPCR according to the 2^-ΔΔC_T method (Livak and Schmittgen, 2001) using E. salsugineum Thhalv10028913m as a reference gene (see Materials and Methods). Expression was normalized to the expression level in dry seeds, which was assigned a value of 1. Data are mean ± SD (n = 3) and are representative of two independent experiments. Asterisks indicate significant differences P < 0.05 (Student’s t-test) between the levels of expression in control and salt-treated seeds at the indicated time points. Dark gray bars, Control; light gray bars, 200 mM NaCl; D, Dry seeds; 0–48, hours after stratification.

FIGURE 8

Removal of the seed coat leads to development of seedlings from excised E. salsugineum embryos. The seed coat with the single layer of endosperm cells was removed from salt-treated E. salsugineum seeds at 2 DAS and embryos were transferred to fresh 200 mM salt-containing medium. (A) Embryo-derived seedlings photographed at 7 DAS. (B) Salt-treated intact seeds transferred to the same fresh 200 mM salt-containing medium at 2 DAS and photographed at 7 DAS were used as negative control. (C) Cotyledon emergence of excised embryos was scored at 7 DAS. Data are mean ± SD of three independent biological experiments. Each experiment contained ca. 20 embryos. DAS, days after stratification.

FIGURE 9

Model of salt-mediated inhibition of E. salsugineum seed germination. Seeds imbibed in the absence of salt exhibit increased GA levels and reduced ABA content. The expression of genes involved in repressing germination such as RGL2, ABI5, and DOG1 is down-regulated, storage reserves are mobilized, seed metabolism associated with germination becomes active, seed desiccation tolerance is lost, and germination occurs. In contrast, salt exposure (response to the osmotic component) causes the opposite process: a decreased GA/ABA ratio and increased expression of germination repressor genes. Storage reserves are not mobilized, and germination-associated metabolic changes are arrested or retarded accompanied by induction of stress tolerance-associated processes. These features are characteristic of a dormancy-like state that is reversible upon alleviation of saline conditions. Increased DOG1 expression in salt-treated seeds may also repress micropylar endosperm cap weakening thereby preventing radicle emergence. Solid red arrows, increase; solid blue arrows, decrease.