Selection for low or high primary dormancy in Lolium rigidum Gaud seeds results in constitutive differences in stress protein expression and peroxidase activity

Abstract

Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0-42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development.

Fig. 1.

Proteomic analysis of low- and high-dormancy seeds. Soluble (A, B) and insoluble (C, D) proteins extracted from low-dormancy (A, C) and high-dormancy (B, D) seeds were separated by 2D-PAGE and their spot patterns compared. Representative gels from four independent replicates are shown. Protein spots with a differential intensity of >3-fold (P <0.05), as determined by scanning densitometry, are circled.

Fig. 2.

Effect of extreme stratification temperatures on germination and viability of low-dormancy (LD) and high-dormancy (HD) seeds. LD and HD seeds were (A) dark stratified for 21 d at 6, 20, or 35 °C and then transferred to standard germination conditions (25/15 °C, 12 h photoperiod) for a further 42 d, or (B) dark stratified for 21 d at 20 °C, then for 21 d at 6, 20, or 35 °C, and finally transferred to germination conditions for a final 21 d, after which time, germination (as a percentage of the total number of seeds) was recorded. The viability of ungerminated seeds was assessed by tetrazolium staining and the number of viable seeds was added to the number of germinated seeds to give total seed viability. Values are means ±SE (n=4). Different letters (lower case for germination, upper case for viability) above columns within each panel indicate significant differences between means; pooled LSD values were 12.7 for germination and 6.3 for viability.

Fig. 3.

Changes during dark stratification of low-dormancy (LD) and high-dormancy (HD) seeds. Seeds were stratified at 20 °C in the dark for 0–42 d and assessed for (A) final germination percentage at 42 d after transfer of stratified seeds to germination conditions, (B) glyoxalase I activity, (C) total peroxidase activity, and (D) the ratio of reduced (GSH) to total glutathione. Parameters in B, C, and D were measured immediately after 0, 7, 21, or 42 d dark stratification. Values are means ±SE (n=4).

Fig. 4.

Correlation between germination and stress defence parameters in seeds. The linear correlation (r²) between (A) germination and total peroxidase (PX) activity, (B) germination and GSSG concentration, (C) PX activity and glutathione reduction state (E_GSSG/2GSH), or (D) GSSG concentration and glyoxalase I activity was assessed using the data from the 0–42 d time course of dark stratification presented in Fig. 3 and Table 3. Enzyme activities are expressed as μmol min⁻¹ mg⁻¹ protein, GSSG concentration as nmol g⁻¹ dry weight, germination as % viable seeds germinated at 42 d, and E_GSSG/2GSH as mV.

Fig. 5.

Effect of hydrogen peroxide and ascorbate on seed germination and glutathione status. Low-dormancy (LD) and high-dormancy (HD) seeds were treated with 10 mM H₂O₂ or ascorbate (pH 6.3) and their (A) germination after 42 d in standard germination conditions, (B) glutathione concentration, and (C) glutathione half-cell reduction potential were assessed. Parameters in B and C were measured in seeds that had been imbibed on the appropriate chemical for 2 d in the dark at 20 °C. Deionized water was used as a control. Values are means ±SE (n=4). Pooled LSD values were: (A) 9.3; (B) 25.1; (C) 12.1.