MtHSFA9 plays a role in the release of dormancy during late maturation
When the viability of the seed lots was tested, we noticed that promptly after harvest, seeds took a very long time to germinate. Since M. truncatula seeds exhibit physiological dormancy, apparent from a strong reduction in germination speed that is released during after-ripening (Bolingue et al, 2010), we decided to further investigate this behaviour. Seeds of the different genotypes were harvested at pod abscission and dried for three days at 43% RH to homogenize the water content before imbibition. Whereas wild type seeds germinated within 2-3 days, seeds from Mthsfa9 lines germinated at around 8-10 days (Fig. 4a). Stratification for 3d at 4°C drastically increased the germination speed of the mutants, and only a very slight difference was still apparent between wild type and Mthsfa9 lines (Fig. 4b). During after-ripening, the difference in germination speed between the mutants and wild type seeds remained significant up until 5 months, but the relative rate of after-ripening did not differ between the genotypes (Fig. 4c).
Since MtHSFA9 transcript levels increased around 20 DAP and reached a maximum at 36 DAP (Fig. S1a), dormancy levels were determined as mean germination time in freshly harvested seeds from 20 DAP until pod abscission (Fig. 4d). At 20 DAP, prior to the acquisition of dormancy (Bolingue, Vu, Leprince & Buitink 2010), no difference was found in the dormancy levels between the genotypes (Fig. S7a). Significant differences were observed at final maturation, with the highest difference prior to full maturation drying, at 40 DAP (Fig. 4d, Fig. S7b). Absence of MtHSFA9 doubled the mean germination time from 35 to 70d, indicating that MtHSFA9 regulates the depth of dormancy acquisition during late maturation. Seed development at 26°C did not change the dormancy level in the Mthsfa9 mutants compared to wild type (Fig. S7c). Freshly harvested Arabidopsis hsfa9 seeds showed reduced germination but this was comparable to wild type seeds (Fig. S7d). After-ripening increased the germination percentage and speed, but no difference could be detected between the Athsfa9 and wild type seeds (Fig. S7d).