Transcriptome changes associated with relief of sly1-2 seed dormancy through after-ripening or overexpression of the gibberellin-receptor GID1b

Seed dormancy is the inability for seeds to germinate even under favorable conditions. In the Arabidopsis Landsberg <i>erecta</i> (L<i>er</i>) ecotype, 2 weeks of dry storage, called after-ripening, is sufficient to relieve seed dormancy. Such seed is referred to as after-ripened (AR) and has a high rate of germination when imbibed. While widespread transcriptome changes have been previously observed with seed dormancy loss, this experiment was designed to characterize transcriptional changes associated with the increased seed dormancy and dormancy loss of the gibberellin (GA) hormone-insensitive <i>sleepy1-2</i> (<i>sly1-2</i>) mutant. The <i>SLY1</i> gene encodes the F-box subunit of an SCF E3 ubiquitin ligase needed for GA-triggered proteolysis of DELLA repressors of seed germination. In the <i>sly1-2</i> mutant, GA-directed DELLA proteolysis cannot occur leading to DELLA protein accumulation and increased dormancy. <i>sly1-2</i> mutant seeds are fully dormant at 2 weeks of dry storage (0% germination), but germinate well with very long after-ripening (51% germination after 19 months). <i>sly1-2</i> seed germination can also be rescued by overexpression of the GA receptor, <i>GA-INSENSITIVE DWARF1b</i> (<i>GID1b-OE</i>), which resulted in 74% germination at 2 weeks of dry storage. In this experiment, we compared seeds of wild-type L<i>er</i> at 2 weeks of dry storage (non-dormant), dormant <i>sly1-2</i> (2 weeks of dry storage; <i>sly1-2</i>(D)), long after-ripened <i>sly1-2</i> (non-dormant, 19 months of dry storage; <i>sly1-2</i>(AR)), and <i>sly1-2 GID1b-OE</i> (non-dormant, 2 weeks of dry storage). Samples were collected at two imbibition timepoints: 1) a 0h timepoint after 4 days at 4°C, and 2) a 12h timepoint after 4 days at 4°C followed by 12 hours in the light at 22°C. These timepoints were selected to capture the transcriptomes at an early and late time in Phase II of imbibition. Using this experimental design we were able to determine transcriptome differences associated with seed dormancy in the <i>sly1-2</i> mutation (L<i>er</i> wt vs <i>sly1-2</i>(D)), and changes associated with <i>sly1-2</i> dormancy loss through dry after-ripening (<i>sly1-2</i>(AR) vs <i>sly1-2</i>(D)) or through <i>GID1b</i>-overexpression (<i>sly1-2 GID1b-OE</i> vs <i>sly1-2</i>(D)). Seeds for L<i>er</i> wt, <i>sly1-2</i>(D), and <i>sly1-2 GID1b-OE</i> were grown alongside each other under the same conditions and after-ripened for 2 weeks. Seeds from <i>sly1-2</i>(AR) were grown under the same conditions in advance of the other lines to allow for the long after-ripening requirement. RNA was extracted using a phenol-chloroform-based extraction from three biological replicates per treatment.

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Nelson SK, Steber CM